IL282610A - Anti-ctla4 antibodies, antibody fragments, their immunoconjugates and uses thereof - Google Patents

Anti-ctla4 antibodies, antibody fragments, their immunoconjugates and uses thereof

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Publication number
IL282610A
IL282610A IL282610A IL28261021A IL282610A IL 282610 A IL282610 A IL 282610A IL 282610 A IL282610 A IL 282610A IL 28261021 A IL28261021 A IL 28261021A IL 282610 A IL282610 A IL 282610A
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Israel
Prior art keywords
antibody
ctla4
immunoconjugate
antibodies
pharmaceutical composition
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IL282610A
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Hebrew (he)
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IL282610B (en
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Jay M Short
Gerhard Frey
Hwai Wen Chang
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Bioatla Inc
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Publication of IL282610A publication Critical patent/IL282610A/en
Publication of IL282610B publication Critical patent/IL282610B/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
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    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68033—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a maytansine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68035—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a pyrrolobenzodiazepine
    • A—HUMAN NECESSITIES
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    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
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    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68037—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a camptothecin [CPT] or derivatives
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    • A—HUMAN NECESSITIES
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    • C—CHEMISTRY; METALLURGY
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57492—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds localized on the membrane of tumor or cancer cells
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    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
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Description

ANTI-CTLA4 ANTIBODIES, ANTIBODY FRAGMENTS, THEIR IMMUNOCONJUGATES AND USES THEREOF FIELD OF THE DISCLOSURE 1. 1. id="p-1" id="p-1" id="p-1" id="p-1" id="p-1" id="p-1" id="p-1" id="p-1" id="p-1" id="p-1" id="p-1" id="p-1"
[0001] This disclosure relates anti-CTLA4 antibodies ,antibody fragments and immunoconjugates of such antibodies and antibody fragments and uses of the antibodies, antibody fragments and immunoconjugates in diagnostic and therapeuti cmethods.
BACKGROUND OF THE DISCLOSURE 2. 2. id="p-2" id="p-2" id="p-2" id="p-2" id="p-2" id="p-2" id="p-2" id="p-2" id="p-2" id="p-2" id="p-2" id="p-2"
[0002] The vertebrate immune system requires multiple signal sto achieve an optimal immune activation; see, e.g., Janeway, Cold Spring Harbor Symp. Ouant. Biol. 54:1-14 (1989); Paul William E., ed. Raven Press, N.Y., Fundamental Immunology, 4th edition (1998), particularl ychapters 12 and 13, pages 411 to 478. Interactions between T lymphocytes (T cell s)and antigen presenting cells (APC) are essential to immune activation.
Levels of many cohesive molecules found on T cells and APCs increase during immune activation (Springer et al., A. Rev. Immunol., 5:223-252 (1987); Shaw and Shimuzu, Current Opinion in Immunology, Eds. Kindt and Long. 1:92-97 (1988)); and Hemler, Immunology Today, 9:109-113 (1988)). Increased level ofs these molecules may help explain why activated APCs are more effective at stimulating antigen-specific T cel lproliferation than are resting APCs (Kaiuchi et al., J. Immunol., 131:109-114 (1983); Kreigeret al., J. Immunol., 135:2937-2945 (1985); McKenzie ,J. Immunol., 141:2907-2911 (1988); and Hawrylowicz and Unanue, J. Immunol., 141:4083-4088 (1988)). 3. 3. id="p-3" id="p-3" id="p-3" id="p-3" id="p-3" id="p-3" id="p-3" id="p-3" id="p-3" id="p-3" id="p-3" id="p-3"
[0003] T cel limmune response is a comple xprocess that involves cell-cel intleractions (Springer et al., A. Rev. Immumol., 5:223-252 (1987)), particularly between T cells and accessory cells such as APCs, as wel las production of solubl ime mune mediators (cytokines or lymphokines) (Dinarell o,New Engl. Jour: Med., 317:940-945 (1987); Sallusto, J. Exp. 179:1109-1118 (1997)). The immune response is regulated by several T-cel lsurface receptors, including the T-cel lreceptor comple x(Weiss, Ann. Rev. Immunol., 4:593-619 (1986)) and other "accessory" surface molecules (Allison, Curr. Opin. Immunol., 6:414-419 (1994); Springer (1987) supra). Many of these accessory molecules are naturally occurring cell surface differentiation antigens defined by the reactivity of monoclona lantibodies on the surface of cells (McMichael Ed.,, Leukocyte Tiping III, Oxford Univ. Press ,Oxford, N.Y. (1987)). 4. 4. id="p-4" id="p-4" id="p-4" id="p-4" id="p-4" id="p-4" id="p-4" id="p-4" id="p-4" id="p-4" id="p-4" id="p-4"
[0004] CTLA4 is a T cell surface molecul thate was originally identified by differential screening of a murine cytolytic T cell cDNA library (Brunet et al., Nature 328:267-270WO 2020/092155 PCT/US2019/058066 (1987)). CTLA4 is also a member of the immunoglobul in(Ig) Superfamily. CTLA4 comprises a singl eextracellular Ig domain. CTLA4 transcripts have been found in T cell populations having cytotoxic activity, suggesting that CTLA4 might function in the cytolytic response (Brunet et al., Supra; Brunet et al., Immunol. Rev., 103:21-36 (1988)). Researchers have reported the cloning and mapping of a gene for the human counterpart of CTLA4 (Dariavach et al., Eur: J. Immumol., 18:1901-1905 (1988)) to the same chromosomal region (2d,33-34) as CD28 (Lafage-Pochitaloffet al., Immuno genetics 31:198-201 (1990)). Sequence comparison between this human CTLA4 DNA and that encoding CD28 proteins reveals significant homology of sequence, with the greatest degree of homology in the juxtamembrane and cytoplasmic regions (Brunet et al., 1988, Supra: Dariavach et al., 1988, Supra). . . id="p-5" id="p-5" id="p-5" id="p-5" id="p-5" id="p-5" id="p-5" id="p-5" id="p-5" id="p-5" id="p-5" id="p-5"
[0005] Some studies have suggeste dthat CTLA4 has an analogous function as a secondary costimulator (Linsle yet al., J. Exp. Med., 176:1595-1604 (1992); Wu et al., J. Exp. Med., 185:1327-1335 (1997) and U.S. Patent Nos. 5,977,318; 5,968,510; 5,885,796; and ,885.579). However, others have reported that CTLA4 has an opposing role as a dampener of T cell activation (Krummel J., Exp. Med., 182:459-465 (1995); Krummel et al., Int'l Immunol., 8:519-523 (1996); Chamber set al., Immunity, 7:885-895 (1997)). It has been reported that CTLA4 deficient mice suffer from massive lymphoproliferati on(Chambers et al., supra). It has also been reported that a CTLA4 blockade augments T cel lresponses in vitro (Walunas et al., Immunity, 1:405-413 (1994)) and in vivo (Kearney, J. Immunol., 155:1032-1036 (1995)), exacerbates antitumor immunity (Leach, Science, 271:1734-1736 (1996)), and enhances an induced autoimmune diseas e(Luhder, J Exp. Med., 187:427-432 (1998)). It has also been reported that CTLA4 has an alternative or additional impact on the initial character of the T cell immune response (Chambers, Curr. Opin. Immunol., 9:396^404 (1997); Bluestone, J. Immunol., 158:1989-1993 (1997); Thompson, Immunity, 7:445-450 (1997)). This is consistent with the observation that some autoimmune patients have autoantibodies to CTLA4. It is possible that CTLA4 blocking antibodies have a pathogenic role in these patients (Matsui, J. Immunol., 162:4328-4335 (1999)). 6. 6. id="p-6" id="p-6" id="p-6" id="p-6" id="p-6" id="p-6" id="p-6" id="p-6" id="p-6" id="p-6" id="p-6" id="p-6"
[0006] CTLA4 has been shown to negatively regulate immune activation through both intrinsic and extrinsic mechanisms. See Grosso and Kunkel Cance, r Immunity, 13: 5 (2013).
Specifically, (i) reverse signalling through CD80 and CD86 on APCs resul tsin suppression of T cell responses and/or promotes conversion of naive T cell tos Tregs, (ii) signaling through CTLA3 stimulates production of regulatory cytokine ssuch as TGF־p, resulting in inhibition of antigen presentation by APCs and inhibition of T cell function, (iii) binding of 2WO 2020/092155 PCT/US2019/058066 CTLA4 to CD80/CD86 reduces availability of these ligands for binding by CD28, resulting in reduced activation of T cell bys APCs, (iv) binding of CTLA4 to CD80/CD86 causes thei r transendocytosis, reducing the ability for APCs to activate T cells, (v) CTLA4 recruits inhibitory proteins such as PP2A and PTPN11 to the T cel lsynapse, inhibiting signalling through CD28 and TCR, (vi) CTLA4 acts as a high affinity competitor occupying CD80/86 and thereby preventing binding by CD28, (vii) a solubl splicee variant of CTLA4 may be capable of inhibiting T cel lactivation, and (viii) CTLA4 inhibits the T cell stop signal, which is important for activation of T cell bys APCs. 7. 7. id="p-7" id="p-7" id="p-7" id="p-7" id="p-7" id="p-7" id="p-7" id="p-7" id="p-7" id="p-7" id="p-7" id="p-7"
[0007] Thus, inhibition of CTLA4 has been shown to promote stimulation of adaptive immune response and T cell activation. CTLA4-blocking antibodies have been shown to be efficacious in mouse models of cancer, and anti-CTLA4 antibodies such as ipilimumab (WO 2001/014424) and tremelimum abare being investigated as strategie sto promote anti-tumor immunity in cancer. Blockade of CTLA4 is also a promising therapeuti cstrategy for disorders associated with T cel lexhaustion such as chronic viral infection. 8. 8. id="p-8" id="p-8" id="p-8" id="p-8" id="p-8" id="p-8" id="p-8" id="p-8" id="p-8" id="p-8" id="p-8" id="p-8"
[0008] Antibodies to CTLA4 have been previously develope d.U.S. Patent No. 9,758,583 disclose antibodies s or antibody fragments that are said to bind to one or both of human and murine CTLA4, which may be formulated into compositions for treatment of cancer. Some of the antibodies or antibody fragments are also said to optionally inhibit or preven tinteraction or functional association between human CTLA4 and human CD80 or CD86, or between murine CTLA4 and murine CD80 or CD86. Such inhibition or prevention of interaction or functional association between CTLA4 and CD80 or CD86 may inhibit or preven tCD80 or CD86-mediated activation of CTLA4, CD80/CTLA4 signalling or CD86/CTLA4 signalling. 9. 9. id="p-9" id="p-9" id="p-9" id="p-9" id="p-9" id="p-9" id="p-9" id="p-9" id="p-9" id="p-9" id="p-9" id="p-9"
[0009] US 2009/0252741 also discloses monoclonal antibodies that bind to human CTLA4.
These anti-CTLA4 antibodies are said to induce protection against cancer and also demonstrate some autoimmune side effects. The antibody that induced the strongest protection against cancer also induced the least autoimmune side effects. US 2009/0252741 also provides a method for selecting optimal anti-CTLA4 antibodies or other therapeuti c agents with the most desirable balance between cancer protection and autoimmune side effects. . . id="p-10" id="p-10" id="p-10" id="p-10" id="p-10" id="p-10" id="p-10" id="p-10" id="p-10" id="p-10" id="p-10" id="p-10"
[0010] US 2016/0237154 disclose comps ositions and methods relating to or derived from anti-CTLA4 antibodies or antibody fragments. The anti-CTLA4 antibodies and antibody fragments may block binding of human CTLA4 to human B7, and thus are said to be suitable for treatment of cancers of the prostate, kidney, colon ,lung or breast ;pathogenic infections; diseases associated with the central nervious system e.g., amyloidogeni cdiseases including 3WO 2020/092155 PCT/US2019/058066 Alzheimer's disease; and diseases with inflammatory or allergic components such as graft versus host disease, host versus graft disease, allergies, autoimmune diseases and other inflammatory diseases. 11. 11. id="p-11" id="p-11" id="p-11" id="p-11" id="p-11" id="p-11" id="p-11" id="p-11" id="p-11" id="p-11" id="p-11" id="p-11"
[0011] Though antibodies against CTLA4 are known and commercially available, it is desirable to find improved anti-CTLA4 antibodies that are suitable for cancer therapy with reduced or minimal side effects. The present invention provides anti-CTLA4 antibodies or antibody fragments that are suitable for therapeuti cand diagnostic use, especiall fory diagnosis and treatment of cancers. Some of thes eanti-CTLA4 antibodies or antibody fragments may have a higher binding affinity to CTLA4 in a tumor in comparison with CTLA4 present in normal tissue. These anti-CTLA4 antibodies or antibody fragments typicall yhave at least comparable efficacy to known anti-CTLA4 antibodies or antibody fragments. In addition, the present anti-CTLA4 antibodies or antibody fragments may exhibit reduced side effects in comparison with monoclonal anti-CTLA4 antibodies known in the art.
These advantages may provide a more selective treatment of the CTLA4 in a tumor and may permi tuse of higher dosages of these anti-CTLA4 antibodies or antibody fragments as a resul oft the selectivi tyfor CTLA4 in a tumor, whereby more effective therapeutic treatments can be realized without a corresponding increase in undesirabl eside effects.
SUMMARY OF THE DISCLOSURE 12. 12. id="p-12" id="p-12" id="p-12" id="p-12" id="p-12" id="p-12" id="p-12" id="p-12" id="p-12" id="p-12" id="p-12" id="p-12"
[0012] In one aspect, the presen tinvention provides isolated heavy chain variable region polypeptides that specifically bind to the CTLA4 protein. These polypeptides include three complementari tydetermining regions having the Hl, H2, and H3 sequences wherein:, the Hl sequence is GFTFSHYTMH (SEQ ID NO: 1); the H2 sequence is FIX1YX2GNX3KX4X5AX6SX7KG (SEQ ID NO: 2); and the H3 sequence is TGWLGPFDX8 (SEQ ID NO: 3); wherein X! is S or D; X2 is D, H or I, X3 is N or Y; X4 is Y or I; X5 is Y or E; X6 is D or K; X7 is V or M; and X8 is Y or I. 13. 13. id="p-13" id="p-13" id="p-13" id="p-13" id="p-13" id="p-13" id="p-13" id="p-13" id="p-13" id="p-13" id="p-13" id="p-13"
[0013] In another aspect, the present invention includes a product formed by a combination of any of the above-described isolated heavy chain variable region polypeptides with an isolated light chain variable region polypeptide selected from isolated light chain variable region polypeptides that include three complementarity determining regions having the LI, L2, and L3 sequences, wherein: the LI sequence is RX9SQX0X1GSSYLA (SEQ ID NO: 4); the L2 sequence is GAFSRATGX12 (SEQ ID NO: 5); and 4WO 2020/092155 PCT/US2019/058066 the L3 sequence is QQDGSSPWT ( SEQ ID NO: 6), wherein Xg is A or I; X!o is Y, S or H; Xu is V or G; X!2 is V or I. 14. 14. id="p-14" id="p-14" id="p-14" id="p-14" id="p-14" id="p-14" id="p-14" id="p-14" id="p-14" id="p-14" id="p-14" id="p-14"
[0014] In each of the previous embodiments the, H2 sequence may be selected from FIDYHGNNKYYADSVKG, FISYDGNNKIYADSVKG, FISYDGNNKYYADSVKG, FISYDGNYKYYADSVKG, FISYDGNYKYYAKSVKG, FISYHGNNKYEADSVKG, FISYHGNNKYYADSVKG, FISYIGNYKYYADSMKG, and FISYIGNYKYYADSVKG. . . id="p-15" id="p-15" id="p-15" id="p-15" id="p-15" id="p-15" id="p-15" id="p-15" id="p-15" id="p-15" id="p-15" id="p-15"
[0015] In each of the previous embodiments the, H3 sequence may be selected from TGWLGPFDY and TGWLGPFDI. 16. 16. id="p-16" id="p-16" id="p-16" id="p-16" id="p-16" id="p-16" id="p-16" id="p-16" id="p-16" id="p-16" id="p-16" id="p-16"
[0016] In each of the previous embodiments the, LI sequence may be selected from RASQHVGSSYLA, RASQSVGSSYLA, RASQYGGSSYLA, RASQYVGSSYLA, and RISQYVGSSYLA. 17. 17. id="p-17" id="p-17" id="p-17" id="p-17" id="p-17" id="p-17" id="p-17" id="p-17" id="p-17" id="p-17" id="p-17" id="p-17"
[0017] In each of the previous embodiments the, L2 sequence may be selected from GAFSRATGI and GAFSRATGV. 18. 18. id="p-18" id="p-18" id="p-18" id="p-18" id="p-18" id="p-18" id="p-18" id="p-18" id="p-18" id="p-18" id="p-18" id="p-18"
[0018] In some embodiments the, isolated heavy chain variable region polypeptide may have a sequence selected from SEQ ID NOS: 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, and 38. In each of thes eembodiments, the isolated light chain variable region polypeptide may have a sequence selected from SEQ ID NOS: 7, 9, 11, 13, 15, 17, 19, 21, 23, ,27, 29, 31, 33,35, and 37. 19. 19. id="p-19" id="p-19" id="p-19" id="p-19" id="p-19" id="p-19" id="p-19" id="p-19" id="p-19" id="p-19" id="p-19" id="p-19"
[0019] In one embodiment the, antibody comprises a light chain variable region polypeptide and a heavy chain variable region polypeptide having a pair of sequences selected from the pairs: SEQ ID NOS: 7 and 8, SEQ ID NOS: 9 and 10, SEQ ID NOS: 11 and 12, SEQ ID NOS: 13 and 14, SEQ ID NOS: 15 and 16, SEQ ID NOS: 17 and 18, SEQ ID NOS: 19 and , SEQ ID NOS: 21 and 22, SEQ ID NOS: 23 and 24, SEQ ID NOS: 25 and 26, SEQ ID NOS: 27 and 28, SEQ ID NOS: 29 and 30, SEQ ID NOS: 31 and 32, SEQ ID NOS: 33 and 34, SEQ ID NOS: 35 and 36 and SEQ ID NOS: 37 ad 38. . . id="p-20" id="p-20" id="p-20" id="p-20" id="p-20" id="p-20" id="p-20" id="p-20" id="p-20" id="p-20" id="p-20" id="p-20"
[0020] In yet another aspect, the present invention provides an anti-CTLA4 antibody or antibody fragment that includes any of the isolated heavy chain variable region polypeptides of the invention described above. 21. 21. id="p-21" id="p-21" id="p-21" id="p-21" id="p-21" id="p-21" id="p-21" id="p-21" id="p-21" id="p-21" id="p-21" id="p-21"
[0021] In yet another aspect, the present invention provides an anti-CTLA4 antibody or antibody fragment that includes a combination of any of the isolated heavy chain variable region polypeptides of the invention described above with any one of the isolated light chain variable region polypeptides of the invention described above. 22. 22. id="p-22" id="p-22" id="p-22" id="p-22" id="p-22" id="p-22" id="p-22" id="p-22" id="p-22" id="p-22" id="p-22" id="p-22"
[0022] In yet another aspect, the present invention provides an immunoconjugate that includes any of the antibody or antibody fragments of the invention described above. In the 5WO 2020/092155 PCT/US2019/058066 immunoconjugate, the antibody or antibody fragment may be conjugated to an agent selected from a chemotherapeutic agent, a radioactive atom, a cytostatic agent and a cytotoxic agent. 23. 23. id="p-23" id="p-23" id="p-23" id="p-23" id="p-23" id="p-23" id="p-23" id="p-23" id="p-23" id="p-23" id="p-23" id="p-23"
[0023] In yet another aspect, the present invention provides a pharmaceutical composition that includes any of the polypeptide s,the antibodies ,the antibody fragments ,and the immunoconjugates of the invention described above, together with a pharmaceutical ly acceptable carrier. A singl edose of the pharmaceutica lcomposition may include an amount of the polypeptide, the antibody, the antibody fragment ,or the immunoconjugate of about 135 mg, about 235 mg, about 335 mg, about 435 mg, about 535 mg, about 635 mg, about 735 mg, about 835 mg, about 935 mg, aboutl035 mg, about 1135 mg, about 1235 mg, or about 1387 mg. 24. 24. id="p-24" id="p-24" id="p-24" id="p-24" id="p-24" id="p-24" id="p-24" id="p-24" id="p-24" id="p-24" id="p-24" id="p-24"
[0024] A singl edose of the pharmaceutical composition of may include an amount of the polypeptide, the antibody, the antibody fragment, or the immunoconjugate in a range of 135- 1387 mg, 135 -235 mg, 235 -335 mg, 335 -435 mg, 435 -535 mg, 535 -635 mg, 635 -735 mg, 735 -835 mg, 835 -935 mg, 935 -1035 mg, 1035 -1135 mg, 1135 -1235 mg, or 1235 -1387 mg. . . id="p-25" id="p-25" id="p-25" id="p-25" id="p-25" id="p-25" id="p-25" id="p-25" id="p-25" id="p-25" id="p-25" id="p-25"
[0025] Each of the foregoing pharmaceutical compositions may further include an immune checkpoint inhibitor molecule that is different from the polypeptide or the antibody or antibody fragment .The immune checkpoint inhibitor molecul maye be an antibody or antibody fragment against an immune checkpoint. The immune checkpoint may be selected from LAG3, TIM3, TIGIT, VISTA, BTLA, OX40, CD40, 4-1BB, PD-1, PD-L1, GITR, B7- H3, B7-H4, KIR, A2aR, CD27, CD70, DR3, and IGOS or the immune checkpoint may be PD-1 orPD-Ll. 26. 26. id="p-26" id="p-26" id="p-26" id="p-26" id="p-26" id="p-26" id="p-26" id="p-26" id="p-26" id="p-26" id="p-26" id="p-26"
[0026] Each of the foregoing pharmaceutica lcompositions may further include an antibody or antibody fragment against an antigen selected from PD1, PD-L1, AXL, ROR2, CD3, HER2, B7-H3, ROR1, SFRP4 and a WNT protein. The WNT protein may be selected from WNT1, WNT2, WNT2B, WNT3, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9B, WNT10A, WNT10B, WNT11 and WNT16. 27. 27. id="p-27" id="p-27" id="p-27" id="p-27" id="p-27" id="p-27" id="p-27" id="p-27" id="p-27" id="p-27" id="p-27" id="p-27"
[0027] In yet another aspect, the present invention provides a kit for diagnosis or treatment including any of the polypeptides, the antibodies ,the antibody fragments, or the immunoconjugates of the present invention described above. 28. 28. id="p-28" id="p-28" id="p-28" id="p-28" id="p-28" id="p-28" id="p-28" id="p-28" id="p-28" id="p-28" id="p-28" id="p-28"
[0028] In yet another aspect, the present invention provides an anti-CTLA4 antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprise sthree complementari tydetermining regions having amino 6WO 2020/092155 PCT/US2019/058066 acid sequence sof SEQ ID NOS:39-41 and the light chain variable region comprises three complementari tydetermining regions having amino acid sequences of SEQ ID NOS:42-44. 29. 29. id="p-29" id="p-29" id="p-29" id="p-29" id="p-29" id="p-29" id="p-29" id="p-29" id="p-29" id="p-29" id="p-29" id="p-29"
[0029] In the previous embodiment, the heavy chain variable region may have an amino acid sequence of SEQ ID NO:8 and the light chain variable region may have an amino acid sequence of SEQ ID NO:7.
BRIEF DESCRIPTION OF THE DRAWINGS . . id="p-30" id="p-30" id="p-30" id="p-30" id="p-30" id="p-30" id="p-30" id="p-30" id="p-30" id="p-30" id="p-30" id="p-30"
[0030] FIG. 1 shows sequence alignment sof exemplary heavy chain variable regions of anti- CTLA4 antibodies of the present invention. 31. 31. id="p-31" id="p-31" id="p-31" id="p-31" id="p-31" id="p-31" id="p-31" id="p-31" id="p-31" id="p-31" id="p-31" id="p-31"
[0031] FIG. 2 shows sequence alignment sof exemplary light chain variable regions of anti- CTLA4 antibodies of the present invention. 32. 32. id="p-32" id="p-32" id="p-32" id="p-32" id="p-32" id="p-32" id="p-32" id="p-32" id="p-32" id="p-32" id="p-32" id="p-32"
[0032] FIG. 3 A shows a comparison of the binding activity to human CTLA4 at pH 6.0 of two of the anti-CTLA4 antibodies of the present invention to Ipilimumab and an Ipilimumab analog (Ipi-analog), as measured by enzyme linked immunosorbent assay (ELISA). 33. 33. id="p-33" id="p-33" id="p-33" id="p-33" id="p-33" id="p-33" id="p-33" id="p-33" id="p-33" id="p-33" id="p-33" id="p-33"
[0033] FIG. 3B shows a comparison of the binding activity to human CTLA4 at pH 7.4 of the two anti-CTLA4 antibodies of the present invention of Fig. 3 A to Ipilimumab and an Ipilimumab analog (Ipi-analog) as measured by ELISA. 34. 34. id="p-34" id="p-34" id="p-34" id="p-34" id="p-34" id="p-34" id="p-34" id="p-34" id="p-34" id="p-34" id="p-34" id="p-34"
[0034] FIG. 4A shows a comparison of the binding activity to cynomolgus CTLA4 at pH 6.0 of the two anti-CTLA4 antibodies of the present invention of Fig. 3A to Ipilimumab and an Ipilimumab analog (Ipi-analog) as measured by ELISA. . . id="p-35" id="p-35" id="p-35" id="p-35" id="p-35" id="p-35" id="p-35" id="p-35" id="p-35" id="p-35" id="p-35" id="p-35"
[0035] FIG. 4B shows a comparison of the binding activity to cynomolgus CTLA4 at pH 7.4 of the two anti-CTLA4 antibodies of the present invention of Fig. 3A to Ipilimumab and an Ipilimumab analog (Ipi-analog) as measured by ELISA. 36. 36. id="p-36" id="p-36" id="p-36" id="p-36" id="p-36" id="p-36" id="p-36" id="p-36" id="p-36" id="p-36" id="p-36" id="p-36"
[0036] FIG. 5 shows a comparison of the pH-dependent binding activity to human CTLA4 of the two anti-CTLA4 antibodies of the present invention of Fig. 3 A to Ipilimumab and an Ipilimumab analog (Ipi-analog). 37. 37. id="p-37" id="p-37" id="p-37" id="p-37" id="p-37" id="p-37" id="p-37" id="p-37" id="p-37" id="p-37" id="p-37" id="p-37"
[0037] FIG. 6A shows a comparison of the half maximal effective concentration (EC50) of, and the binding activity to human CTLA4 at pH 6.0 of the two anti-CTLA4 antibodies of the present invention of Fig. 3A to Ipilimumab and an Ipilimumab analog (Ipi-analog) as measured by fluorescence-activated cell sorting (FACS) using CHO cells. 38. 38. id="p-38" id="p-38" id="p-38" id="p-38" id="p-38" id="p-38" id="p-38" id="p-38" id="p-38" id="p-38" id="p-38" id="p-38"
[0038] FIG. 6B shows a comparison of the (EC50) of, and the binding activity to human CTLA4 at pH 7.4 of the two anti-CTLA4 antibodies of the present invention of Fig. 3A to Ipilimumab and an Ipilimumab analog (Ipi-analog) as measured by FACS using CHO cells. 7WO 2020/092155 PCT/US2019/058066 39. 39. id="p-39" id="p-39" id="p-39" id="p-39" id="p-39" id="p-39" id="p-39" id="p-39" id="p-39" id="p-39" id="p-39" id="p-39"
[0039] FIG. 7 A shows a comparison of the (EC50) of, and the binding activity to cynomolgus CTLA4 at pH 6.0 of the two anti-CTLA4 antibodies of the present invention of Fig. 3A to Ipilimumab and an Ipilimumab analog (Ipi-analog) as measured by fluorescence-activated cell sorting (FACS) using CHO cells. 40. 40. id="p-40" id="p-40" id="p-40" id="p-40" id="p-40" id="p-40" id="p-40" id="p-40" id="p-40" id="p-40" id="p-40" id="p-40"
[0040] FIG. 7B shows a comparison of the (EC50) of, and the binding activity to cynomolgus CTLA4 at pH 7.4 of the two anti-CTLA4 antibodies of the present invention of Fig. 3A to Ipilimumab and an Ipilimumab analog (ipi-analog) as measured by FACS using CHO cell s. 41. 41. id="p-41" id="p-41" id="p-41" id="p-41" id="p-41" id="p-41" id="p-41" id="p-41" id="p-41" id="p-41" id="p-41" id="p-41"
[0041] FIG. 8A shows a comparison of the (EC50) of, and the saturation of human CTLA4 at pH 7.4 by the anti-CTLA4 antibodies of the presen tinvention of Fig. 3A to the Ipi analog as measured by FACS. 42. 42. id="p-42" id="p-42" id="p-42" id="p-42" id="p-42" id="p-42" id="p-42" id="p-42" id="p-42" id="p-42" id="p-42" id="p-42"
[0042] FIG. 8B shows a comparison of the (EC50) of, and the saturation of cynomolgus CTLA4 at pH 7.4 by the anti-CTLA4 antibodies of the present invention of Fig. 3A to the Ipi analog as measured by FACS. 43. 43. id="p-43" id="p-43" id="p-43" id="p-43" id="p-43" id="p-43" id="p-43" id="p-43" id="p-43" id="p-43" id="p-43" id="p-43"
[0043] FIGS. 9A-9F show binding activity to human CTLA4 of the anti-CTLA4 antibodies of the present invention of Fig. 3 A at pH 6.0 or pH 7.4 as well as at pH 6.0 or pH 7.4 in the presence of different buffers, as measured by ELISA. 44. 44. id="p-44" id="p-44" id="p-44" id="p-44" id="p-44" id="p-44" id="p-44" id="p-44" id="p-44" id="p-44" id="p-44" id="p-44"
[0044] FIGS. 10A-10F show binding activity to human CTLA4 of the anti-CTLA4 antibodies of the present invention of Fig. 3 A at pH 6.0 or pH 7.4 as wel las at pH 6.0 or pH 7.4 in the presenc eof different buffers, as measured by FACS. 45. 45. id="p-45" id="p-45" id="p-45" id="p-45" id="p-45" id="p-45" id="p-45" id="p-45" id="p-45" id="p-45" id="p-45" id="p-45"
[0045] FIG. 11A shows a comparision of the activity the anti-CTLA4 antibodies of the present invention of Fig. 3A to Ipilimumab, an Ipilimumab analog (ipi-analog), an IgG control and a Nivo analog in blocking IL2 secretion in peripheral blood mononuclear cel l (PBMC) cultures at pH 6.2. 46. 46. id="p-46" id="p-46" id="p-46" id="p-46" id="p-46" id="p-46" id="p-46" id="p-46" id="p-46" id="p-46" id="p-46" id="p-46"
[0046] FIG. 1 IB shows a comparision of the activity the anti-CTLA4 antibodies of the present invention of Fig. 3A to Ipilimumab, an Ipilimumab analog (ipi-analog), an IgG control and a Nivo analog in blocking IL2 secretion in PBMC cultures at pH 7.4. 47. 47. id="p-47" id="p-47" id="p-47" id="p-47" id="p-47" id="p-47" id="p-47" id="p-47" id="p-47" id="p-47" id="p-47" id="p-47"
[0047] FIG. 12A shows a comparision of the ECs0 and the activity the anti-CTLA4 antibodies of the present invention of Fig. 3 A to Ipilimumab, an Ipilimumab analog (ipi- analog), and an IgG control in blocking the interaction between CTLA4 and its ligands at pH 6.0. 48. 48. id="p-48" id="p-48" id="p-48" id="p-48" id="p-48" id="p-48" id="p-48" id="p-48" id="p-48" id="p-48" id="p-48" id="p-48"
[0048] FIG. 12B shows a comparision of the EC50 and the activity the anti-CTLA4 antibodies of the present invention of Fig. 3 A to Ipilimumab, an Ipilimumab analog (ipi-analog), and an IgG control in blocking the interaction between CTLA4 and its ligands at pH 7.4. 8WO 2020/092155 PCT/US2019/058066 49. 49. id="p-49" id="p-49" id="p-49" id="p-49" id="p-49" id="p-49" id="p-49" id="p-49" id="p-49" id="p-49" id="p-49" id="p-49"
[0049] FIG. 13A shows a comparison of the EC50 and the activity the anti-CTLA4 antibodies of the present invention of Fig. 3 A to Ipilimumab, an Ipilimumab analog (ipi-analog), and an IgG control in competitive binding to human CTLA4 as a function of the concentration of the ligand hB7-l (hCD80) of CTLA4, as measured by FACS. 50. 50. id="p-50" id="p-50" id="p-50" id="p-50" id="p-50" id="p-50" id="p-50" id="p-50" id="p-50" id="p-50" id="p-50" id="p-50"
[0050] FIG. 13B shows a comparison of the EC50 and the activity the anti-CTLA4 antibodies of the present invention of Fig. 3 A to Ipilimumab, an Ipilimumab analog (ipi-analog), and an IgG control in competitive binding to human CTLA4 as a function of the concentration of the ligand hB7-2 (hCD86) of CTLA4, as measured by FACS. 51. 51. id="p-51" id="p-51" id="p-51" id="p-51" id="p-51" id="p-51" id="p-51" id="p-51" id="p-51" id="p-51" id="p-51" id="p-51"
[0051] FIG. 14A shows a comparison of the activity the anti-CTLA4 antibodies of the present invention of Fig. 3A to Ipilimumab, an Ipilimumab analog (ipi-analog), and an IgG control in competitive binding to human CTLA4 at fixed concentration of ligand hB7-l of CTLA4 as a function of antibody concentration as measured by FACS. 52. 52. id="p-52" id="p-52" id="p-52" id="p-52" id="p-52" id="p-52" id="p-52" id="p-52" id="p-52" id="p-52" id="p-52" id="p-52"
[0052] FIG. 14B shows a comparison of the activity the anti-CTLA4 antibodies of the present invention of Fig. 3A to Ipilimumab, an Ipilimumab analog (ipi-analog), and an IgG control in competitive binding to human CTLA4 at fixed concentration of ligand hB7-2 of CTLA4 as a function of antibody concentration as measured by FACS.
DEFINITIONS 53. 53. id="p-53" id="p-53" id="p-53" id="p-53" id="p-53" id="p-53" id="p-53" id="p-53" id="p-53" id="p-53" id="p-53" id="p-53"
[0053] In order to facilitate understanding of the examples provided herein, certain frequently occurring terms are defined herein. 54. 54. id="p-54" id="p-54" id="p-54" id="p-54" id="p-54" id="p-54" id="p-54" id="p-54" id="p-54" id="p-54" id="p-54" id="p-54"
[0054] In connection with a measured quantity, the term "about" as used herei nrefers to the normal variation in that measured quantity that would be expected by a skill edperson making the measuremen andt exercising a level of care commensurate with the objective of the measuremen andt the precision of the measuring equipment used. Unless otherwise indicated, "about" refers to a variation of +/- 10% of the value provided. 55. 55. id="p-55" id="p-55" id="p-55" id="p-55" id="p-55" id="p-55" id="p-55" id="p-55" id="p-55" id="p-55" id="p-55" id="p-55"
[0055] The term "affinity" as used herein refers to the strength of the sum total of noncovalent interactions between a singl ebinding site of a molecul (e.g.,e an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, "binding affinity" refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecul Xe for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described in the following. 9WO 2020/092155 PCT/US2019/058066 56. 56. id="p-56" id="p-56" id="p-56" id="p-56" id="p-56" id="p-56" id="p-56" id="p-56" id="p-56" id="p-56" id="p-56" id="p-56"
[0056] The term "affinity matured" antibody as used herei nrefers to an antibody with one or more alterations in one or more complimentary determining regions, compared to a parent antibody which does not possess such alterations, such alterations resulting in an improvement in the affinity of the antibody for antigen. 57. 57. id="p-57" id="p-57" id="p-57" id="p-57" id="p-57" id="p-57" id="p-57" id="p-57" id="p-57" id="p-57" id="p-57" id="p-57"
[0057] The term "amino acid" as used herein refers to any organic compound that contains an amino group (-NH2) and a carboxyl group (—COOH); preferably either as free groups or alternativel aftey r condensation as part of peptide bonds. The "twenty naturally encoded polypeptide-forming alpha-amino acids" are understood in the art and refer to: alanine (ala or A), arginine (arg or R), asparagine (asn or N), aspartic acid (asp or D), cysteine (cys or C), gluatamic acid (glu or E), glutamine (gin or Q), glycine (gly or G), histidine (his or H), isoleucine (ile or I), leucine (leu or L), lysin e(lys or K), methionine (me tor M), phenylalani ne(phe or F), proline (pro or P), serine (ser or S), threonine (thr or T), tryptophan (tip or W), tyrosine (tyr or Y), and valine (val or V). 58. 58. id="p-58" id="p-58" id="p-58" id="p-58" id="p-58" id="p-58" id="p-58" id="p-58" id="p-58" id="p-58" id="p-58" id="p-58"
[0058] The term "antibody" as used herei nrefers to intact immunoglobulin molecules, as well as fragments of immunoglobul inmolecules, such as Fab, Fab', (Fab')2, Fv, and SCA fragments, that are capable of binding to an epitope of an antigen. These antibody fragments, which retain some ability to selectively bind to an antigen (e.g., a polypeptide antigen) of the antibody from which they are derived, can be made using wel lknown methods in the art (see, e.g., Harlow and Lane, supra), and are described further, as follows. Antibodies can be used to isolate preparative quantities of the antigen by immunoaffinity chromatography. Various other uses of such antibodies are to diagnose and/or stage diseas e(e.g., neoplasia) and for therapeuti capplication to treat disease, such as for example: neoplasia, autoimmune disease, AIDS, cardiovascular disease, infections, and the like. Chimeric, human-like, humanized or fully human antibodies are particularl yuseful for administration to human patients. 59. 59. id="p-59" id="p-59" id="p-59" id="p-59" id="p-59" id="p-59" id="p-59" id="p-59" id="p-59" id="p-59" id="p-59" id="p-59"
[0059] An Fab fragment consists of a monovalen antit gen-binding fragment of an antibody molecule, and can be produced by digestion of a whol eantibody molecul wite h the enzyme papain, to yield a fragment consisting of an intact light chain and a portion of a heavy chain. 60. 60. id="p-60" id="p-60" id="p-60" id="p-60" id="p-60" id="p-60" id="p-60" id="p-60" id="p-60" id="p-60" id="p-60" id="p-60"
[0060] An Fab' fragment of an antibody molecule can be obtained by treating a whole antibody molecule with pepsin, followed by reduction, to yield a molecule consisting of an intact light chain and a portion of a heavy chain. Two Fab' fragments are obtained per antibody molecule treated in this manner. 61. 61. id="p-61" id="p-61" id="p-61" id="p-61" id="p-61" id="p-61" id="p-61" id="p-61" id="p-61" id="p-61" id="p-61" id="p-61"
[0061] An (Fab')2 fragment of an antibody can be obtained by treating a whol eantibody molecul withe the enzyme pepsin, without subsequent reduction. A (Fab')2 fragment is a dimer of two Fab' fragments ,held together by two disulfide bonds. 10WO 2020/092155 PCT/US2019/058066 62. 62. id="p-62" id="p-62" id="p-62" id="p-62" id="p-62" id="p-62" id="p-62" id="p-62" id="p-62" id="p-62" id="p-62" id="p-62"
[0062] An Fv fragment is defined as a genetically engineered fragment containing the variable region of a light chain and the variable region of a heavy chain expressed as two chains. 63. 63. id="p-63" id="p-63" id="p-63" id="p-63" id="p-63" id="p-63" id="p-63" id="p-63" id="p-63" id="p-63" id="p-63" id="p-63"
[0063] The term "antibody fragment" as used herei nrefers to a molecul othere than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limite dto Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies ;linea rantibodies; single-chai antin body molecules (e.g. scFv); and multispecif icantibodies formed from antibody fragments. 64. 64. id="p-64" id="p-64" id="p-64" id="p-64" id="p-64" id="p-64" id="p-64" id="p-64" id="p-64" id="p-64" id="p-64" id="p-64"
[0064] The terms "anti-CTLA4 antibody," "CTLA4 antibody" and "an antibody that binds to CTLA4" as used herei nrefe rto an antibody that is capable of binding CTLA4 with sufficient affinity such that the antibody is usefu las a diagnostic and/or therapeutic agent in targeting CTLA4. In one embodiment ,the extent of binding of an anti-CTLA4 antibody to an unrelated, non-CTLA4 protein is less than about 10% of the binding of the antibody to CTLA4 as measured, e.g., by a radioimmunoassay (RIA). In certain embodiments, an antibody that binds to CTLA4 has a dissociation constant (Kd) of pM, ^100 nM, ^10 nM, ^1 nM, ^0.1 nM, ^0.01 nM, or ^0.001 nM (e.g. 108־M or less e.g., from 108־M to 1013־M, e.g., from 109־M to 10-13 M). In certain embodiments an, anti-CTLA4 antibody binds to an epitope of CTLA4 that is conserved among CTLA4 from different species. 65. 65. id="p-65" id="p-65" id="p-65" id="p-65" id="p-65" id="p-65" id="p-65" id="p-65" id="p-65" id="p-65" id="p-65" id="p-65"
[0065] The term "binding" as used herei nrefers to interaction of the variable region or an Fv of an antibody with an antigen with the interaction depending upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the antigen. For example, an antibody variable region or Fv recognizes and binds to a specific protein structure rathe rthan to proteins generally. As used herein, the term "specificall bindiny g" or "binding specificall y" means that an antibody variable region or Fv binds to or associates with more frequently, more rapidly, with greater duration and/or with greater affinity with a particular antigen than with other proteins. For example, an antibody variable region or Fv specificall bindsy to its antigen with greater affinity, avidity, more readily, and/or with greater duration than it binds to other antigens. For another example, an antibody variable region or Fv binds to a cell surface protein (antigen) with materially greater affinity than it does to relate dproteins or other cell surface proteins or to antigens commonly recognized by polyreactive natural antibodies (i.e., by naturally occurring antibodies known to bind a variety of antigens naturall yfound in humans). However, "specifically binding" does not necessarily require exclusive binding or non-detectable binding of another antigen, this is meant by the term "selective binding". In one example, "specific binding" of an antibody variable region or Fv 11WO 2020/092155 PCT/US2019/058066 (or other binding region) binds to an antigen, means that the an antibody variable region or Fv binds to the antigen with an equilibrium constant (KD) of 100 nM or les s,such as 50nM or less for, exampl e20nM or less such, as, 15nM or les s,or 10 nM or less ,or 5nM or les s,2 nM or less or, 1 nM or less. 66. 66. id="p-66" id="p-66" id="p-66" id="p-66" id="p-66" id="p-66" id="p-66" id="p-66" id="p-66" id="p-66" id="p-66" id="p-66"
[0066] The terms "cancer" and "cancerous" as used herei nrefe rto or describe the physiological condition in mammal sthat is typicall ycharacterize dby unregulated cel l growth/proliferation. Examples of cancer include, but are not limite dto, carcinoma, lymphoma (e.g., Hodgkin's and non-Hodgkin's lymphoma), blastoma sarcoma, ,and leukem ia.More particular examples of such cancers include squamous cel lcancer, small-cel l lung cancer, non-small cel llung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellula cancr er, gastrointestinal cancer, pancreatic cancer, glioma ,cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma ,breast cancer, colon cancer, colorectal cancer, endometria lor uterine carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, leukem andia other lymphoproliferative disorders, and various types of head and neck cancer. 67. 67. id="p-67" id="p-67" id="p-67" id="p-67" id="p-67" id="p-67" id="p-67" id="p-67" id="p-67" id="p-67" id="p-67" id="p-67"
[0067] The terms "cel lproliferative disorder" and "proliferativ edisorder" as used herein refe rto disorders that are associated with some degree of abnormal cel lproliferation. In one embodiment the, cel lproliferative disorder is cancer. 68. 68. id="p-68" id="p-68" id="p-68" id="p-68" id="p-68" id="p-68" id="p-68" id="p-68" id="p-68" id="p-68" id="p-68" id="p-68"
[0068] The term "chemotherapeutic agent" as used herein refers to a chemica lcompound usefu lin the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphami de(CYTOXAN®); alkyl sulfonate ssuch as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelami nesincluding altretamine , triethylenemelam ine,triethylenephosphoramide, triethylenethiophosphoramid ande trimethylomelamine ace; togenins (especial lybullataci nand bullatacinone); delta-9- tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone; lapachol colc; hicines; betulinic acid; a camptothecin (including the syntheti canalogue topotecan (HYCAMTIN®), CRT-11 (irinotecan, CAMPTOSAR®), acetylcamptotheci n,scopolectin, and 9- aminocamptothecin); bryostatin; callystatin; CC-1065 (including its adozelesin, carzeles in and bizeles insynthetic analogues );podophyllotoxin; podophyllinic acid; teniposide; cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues KW-218, 9 and CB1-TM1); eleutherobi n;pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambuci l,chlornaphazine. 12WO 2020/092155 PCT/US2019/058066 chlorophosphamide, estramustine, ifosfamide, mechlorethamin meche, lorethami oxidene hydrochloride mel, phalan, novembichin ,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin ,fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammall and calicheamici omegan ll (see, e.g., Nicolaou et al., Angew. Chern.
Inti. Ed. Engl., 33: 183-186 (1994)); CDP323, an oral alpha-4 integrin inhibitor; dynemicin, including dynemicin A; an esperamicin; as wel las neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores ),aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including ADRIAMYCIN®, morpholino-doxorubicin, cyanomorpholino- doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin HC1 liposome injection (DOXIL®), liposomal doxorubicin TEC D-99 (MYOCET®), peglylated liposomal doxorubicin (CAELYX®), and deoxy doxorubicin), epirubicin, esorubicin ,idarubicin, marcellomyci n, mitomycins such as mitomycin C, mycophenoli acic d, nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex ,zinostatin, zorubicin; anti-metabolites such as methotrexate, gemcitabine (GEMZAR®), tegafur (UFTORAL@), capecitabine (XELODA®), an epothilone, and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti- adrenal ssuch as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside ami; nolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene ;edatraxate; defofamine; demecolcine; diaziquone; elformithin e;elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol ;nitraerine; pentostatin; phenamet pira; rubicin; losoxantrone; 2- ethylhydrazide procarbaz; ine; PSK® polysaccharide comple x(JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2'-trichlorotriethylami ne;trichothecenes (especiall T-2y toxin, verracurin A, roridin A and anguidine); urethan; vindesine (ELDISINE®, FILDES IN®); dacarbazine; mannomustine; mitobronitol; mitolactol pipobr; oman; gacytosine ;arabinoside ("Ara-C"); 13WO 2020/092155 PCT/US2019/058066 thiotepa; taxoid, e.g., paclitaxel (TAXOL®), albumin-engineere nanopartd icle formulation of paclitaxel (ABRAXANETM), and docetaxel (TAXOTERE®); chloranbucil 6-thioguani; ne; mercaptopurine; methotrexate; platinum agents such as cisplatin, oxaliplatin (e.g., ELOXATIN®), and carboplatin; vincas, which prevent tubulin polymerization from forming microtubules, including vinblastine (VELBAN®), vincristine (ONCOVIN®), vindesine (ELDISINE®, FILDESIN®), and vinorelbine (NAVELBINE®); etoposide (VP-16); ifosfamide; mitoxantrone; leucovorin; novantrone; edatrexate; daunomycin; aminopterin; ibandronate; topoisomeras einhibitor RES 2000; difluoromethylornithine (DMF®); retinoids such as retinoic acid, including bexarotene (TARGRETIN®); bisphosphonates such as clodronate (for example, BONEFOS® or OSTAC®), etidronate (DIDROCAL®), NE-58095, zoledronic acid/zoledronate (ZOMETA®), alendronate (FOSAMAX®), pamidronate (AREDIA®), tiludronate (SKELID®), or risedronate (ACTONEL®); troxacitabine (a 1,3- dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those that inhibit expression of genes in signaling pathways implicate din aberrant cel lproliferation, such as, for example, PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor (EGF- R); vaccines such as THERATOPE® vaccine and gene therapy vaccines, for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; topoisomerase 1 inhibitor (e.g., LURTOTECAN®); rmRH (e.g., ABARELIX@); BAY439006 (sorafenib; Bayer); SU-11248 (sunitinib, SUTENT®, Pfizer); perifosine, COX-2 inhibitor (e.g. celecoxib or etoricoxib), proteosome inhibitor (e.g. PS341); bortezomib (VELCADE®); CCI-779; tipifamib (RI 1577); orafenib, ABTS 10; Bcl-2 inhibitor such as oblimersen sodium (GENASENSE®); pixantrone; EGER inhibitors (see definition below); tyrosine kinase inhibitors (see definition below); serine-threonine kinase inhibitors such as rapamycin (sirolimus RAPA, MUNE®); farnesyltransferase inhibitors such as lonafarnib (SCH 6636, SARAS AR™); and pharmaceutical lyacceptable salts, acids or derivatives of any of the above; as wel las combinations of two or more of the above such as CHOP, an abbreviation for a combined therapy of cyclophosphamide doxorubicin,, vincristine, and prednisolone; and FOLFOX, an abbreviation for a treatment regimen with oxaliplati n(ELOXATIN™) combined with 5-FU and leucovorin. 69. 69. id="p-69" id="p-69" id="p-69" id="p-69" id="p-69" id="p-69" id="p-69" id="p-69" id="p-69" id="p-69" id="p-69" id="p-69"
[0069] Chemotherapeuti agentsc as defined herei ninclude "anti-hormona lagents" or "endocrine therapeutics," which act to regulate, reduce, block, or inhibit the effects of hormones that can promote the growth of cancer. They may be hormones themselves , including, but not limited to: anti-estrogens with mixed agonist/antagonis tprofile, including, tamoxifen (NOLVADEX®), 4-hydroxytamoxifen, toremifene (FARESTON®), idoxifene, 14WO 2020/092155 PCT/US2019/058066 droloxifene, raloxifene (EVISTA®), trioxifene, keoxifene, and selective estrogen receptor modulators (SERMs) such as SERM3; pure anti-estrogens without agonist properties, such as fulvestrant (FASLODEX®), and EM800 (such agents may block estrogen receptor (ER) dimerization, inhibit DNA binding, increase ER turnover, and/or suppres sER levels); aromatase inhibitors, including steroidal aromatase inhibitors such as formestane and exemestane (AROMASIN®), and nonsteroidal aromatase inhibitors such as anastrazole (ARIMIDEX®), letrozole (FEMARA®) and aminoglutethimide, and other aromatase inhibitors include vorozole (RIVISOR®), megestrol acetate (MEGASE®), fadrozole, and 4(5)-imidazoles; lutenizing hormone-releasei hormoneng agonists, including leuprolide (LUPRON® and ELIGARD®), goserelin, buserelin, and tripterelin; sex steroids, including progestines such as megestrol acetate and medroxyprogesterone acetate, estrogens such as diethylstilbestrol and premarin, and androgens/retinoids such as fluoxymesterone, all transretionic acid and fenretinide; onapristone; anti-progesterones; estrogen receptor down- regulators (ERDs); anti-androgens such as flutamide ,nilutamide and bicalutamide; and pharmaceuticall accy eptable salts, acids or derivatives of any of the above; as well as combinations of two or more of the above. 70. 70. id="p-70" id="p-70" id="p-70" id="p-70" id="p-70" id="p-70" id="p-70" id="p-70" id="p-70" id="p-70" id="p-70" id="p-70"
[0070] The term "chimeric" antibody as used herei nrefers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species. 71. 71. id="p-71" id="p-71" id="p-71" id="p-71" id="p-71" id="p-71" id="p-71" id="p-71" id="p-71" id="p-71" id="p-71" id="p-71"
[0071] The term "class" of an antibody as used herei nrefers to the type of constant domain or constant region possesse byd its heavy chain. There are five major classes of antibodies : IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGi, IgG2, IgG3, IgG4, IgAi, and IgA2. The heavy chain constant domains that correspond to the different classe ofs immunoglobulins are called a, 5, 8, y, and p, respectively. 72. 72. id="p-72" id="p-72" id="p-72" id="p-72" id="p-72" id="p-72" id="p-72" id="p-72" id="p-72" id="p-72" id="p-72" id="p-72"
[0072] The term "conditionall yactive antibody" as used herei nrefers to an antibody which is more active under a condition in the tumor microenvironment compared to under a condition in the non-tumor microenvironment. The conditions in the tumor microenvironment include lower pH, higher concentrations of lactate and pyruvate, hypoxia, lower concentration of glucose, and slightl highery temperature in comparison with non-tumor microenvironment.
For example, a conditionally active antibody is virtuall yinactive at normal body temperature , but is active at a higher temperatur ein a tumor microenvironmen t.In yet another aspect, the conditionall yactive antibody is less active in normal oxygenated blood ,but more active under a less oxygenated environment exists in tumor. In yet another aspect, the conditionall y 15WO 2020/092155 PCT/US2019/058066 active antibody is less active in normal physiologic alpH 7.2-7.8, but more active under an acidic pH 5.8-7.0, or 6.0-6.8 that exists in a tumor microenvironment. There are other conditions in the tumor microenvironment know to a person skill edin the field may also be used as the condition in the present invention under which the anti-CTLA4 antibodies to have different binding affinity to CTLA4. 73. 73. id="p-73" id="p-73" id="p-73" id="p-73" id="p-73" id="p-73" id="p-73" id="p-73" id="p-73" id="p-73" id="p-73" id="p-73"
[0073] The term "constitutive" as used herein, as for exampl eapplied to CTLA4 activity, refers to continuous signaling activity of the receptor kinase that is not dependent on the presence of a ligand or other activating molecules. Depending on the nature of the receptor kinase, all of the activity may be constitutive or the activity of the receptor may be further activated by the binding of other molecules (e.g. ligands) .Cellular events that lead to activation of receptor kinase are wel lknown among those of ordinary skill in the art. For example, activation may include oligomerization, e.g., dimerization, trimerization, etc., into higher order receptor complexes Compl. exes may comprise a singl especies of protein, i.e., a homomeric complex. Alternatively compl, exes may comprise at least two different protein species, i.e., a heteromeri complec x. Complex formation may be caused by, for example, overexpression of normal or mutant forms of receptor on the surface of a cell. Complex formation may also be caused by a specific mutation or mutations in a receptor. 74. 74. id="p-74" id="p-74" id="p-74" id="p-74" id="p-74" id="p-74" id="p-74" id="p-74" id="p-74" id="p-74" id="p-74" id="p-74"
[0074] The term "cytostatic agent" as used herein refers to a compound or composition which arrests growth of a cel leithe rin vitro or in vivo. Thus, a cytostatic agent may be one which significantly reduces the percentage of cells in S phase. Furthe rexamples of cytostatic agents include agents that block cel lcycle progression by inducing G0/G1 arrest or M-phase arrest .The humanize danti-Her2 antibody trastuzumab (HERCEPTIN®) is an example of a cytostatic agent that induces G0/G1 arrest. Classical M-phase blockers include the vincas (vincristine and vinblastine), taxanes, and topoisomeras eII inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide ,and bleomycin. Certain agents that arres tG1 also spill over into S-phase arrest ,for example, DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine cispl, atin, methotrexate, 5-fluorouracil, and ara-C. Further information can be found in Mendelsohn and Israel, eds., The Molecular Basis of Cancer, Chapter 1, entitle d"Cell cycle regulation, oncogenes, and antineoplasti cdrugs" by Murakami et al. (W.B. Saunders, Philadelphia, 1995), e.g., p. 13. The taxanes (paclitaxel and docetaxel ) are anticancer drugs both derived from the yew tree. Docetaxel (TAXOTERE®, Rhone- Poulen cRorer), derived from the European yew, is a semisynthet icanalogue of paclitaxel (TAXOL®, Bristol-Myers Squibb). Paclitaxel and docetaxel promote the assembly of 16WO 2020/092155 PCT/US2019/058066 microtubules from tubulin dimers and stabiliz emicrotubule sby preventing depolymerization, which results in the inhibition of mitosis in cells. 75. 75. id="p-75" id="p-75" id="p-75" id="p-75" id="p-75" id="p-75" id="p-75" id="p-75" id="p-75" id="p-75" id="p-75" id="p-75"
[0075] The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents a cellula functir on and/or causes cel ldeath or destruction. Cytotoxic agents include, but are not limite dto radioactive isotopes (e.g., At211,1131,1125, Y90, Re186, Re188, Sm153, Bi212, p32 p^212 anj ra(jioactive isotopes of Lu); chemotherapeuti agenc ts or drugs (e.g., methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambuci l,daunorubicin or other intercalating agents); growth inhibitory agents ;enzymes and fragments thereof such as nucleolyti enzymes;c antibiotics; toxins such as smal moll ecul toxinse or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof; and the various antitumor or anticancer agents disclose below.d 76. 76. id="p-76" id="p-76" id="p-76" id="p-76" id="p-76" id="p-76" id="p-76" id="p-76" id="p-76" id="p-76" id="p-76" id="p-76"
[0076] The term "diabodies" as used herei nrefers to smal antibodyl fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (Vh) connected to a light-chain variable domain (Vl) in the same polypeptide chain (Vh-Vl). By using a linke thatr is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. 77. 77. id="p-77" id="p-77" id="p-77" id="p-77" id="p-77" id="p-77" id="p-77" id="p-77" id="p-77" id="p-77" id="p-77" id="p-77"
[0077] The term "delectably label" as used herei nrefers to any substance whose detection or measurement eit, her directly or indirectly, by physical or chemical means, is indicative of the presence of the CTCs in a sample Repr. esentativ eexamples of useful detectabl elabels, include, but are not limite dto the following: molecules or ions directly or indirectly detectable based on light absorbance, fluorescence refl, ectance, light scatter, phosphorescence, or luminescenc properte ies; molecules or ions detectabl eby thei r radioactive properties; molecules or ions detectable by their nuclear magnetic resonance or paramagnetic properties .Included among the group of molecules indirectly detectable based on light absorbance or fluorescence, for example, are various enzymes which cause appropriate substrate sto convert, e.g., from non-light absorbing to light absorbing molecules, or from non-fluorescent to fluorescent molecules. 78. 78. id="p-78" id="p-78" id="p-78" id="p-78" id="p-78" id="p-78" id="p-78" id="p-78" id="p-78" id="p-78" id="p-78" id="p-78"
[0078] The term "diagnostics" as used herein refers to determination of a subject's susceptibilit toy a diseas eor disorder, determination as to whether a subjec tis presently affected by a diseas eor disorder, prognosis of a subject affected by a diseas eor disorder (e. g., identification of pre- metastatic or metastatic cancerous states ,stages of cancer, or responsivenes ofs cancer to therapy), and therametrics (e. g., monitoring a subject's condition 17WO 2020/092155 PCT/US2019/058066 to provide information as to the effect or efficacy of therapy). In some embodiments, the diagnostic method of this invention is particularly usefu lin detecting early stage cancers. 79. 79. id="p-79" id="p-79" id="p-79" id="p-79" id="p-79" id="p-79" id="p-79" id="p-79" id="p-79" id="p-79" id="p-79" id="p-79"
[0079] The term "diagnostic agent" as used herein refers to a molecul whice h can be directly or indirectly detected and is used for diagnostic purposes. The diagnostic agent may be administered to a subject or a sample The. diagnostic agent can be provided per se or may be conjugated to a vehicle such as a conditionally active antibody. 80. 80. id="p-80" id="p-80" id="p-80" id="p-80" id="p-80" id="p-80" id="p-80" id="p-80" id="p-80" id="p-80" id="p-80" id="p-80"
[0080] The term "effector functions" as used herein refer to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: Clq binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediate cytotoxicityd (ADCC); phagocytosis; down regulation of cel lsurface receptors (e.g. B cell receptor); and B cel l activation. 81. 81. id="p-81" id="p-81" id="p-81" id="p-81" id="p-81" id="p-81" id="p-81" id="p-81" id="p-81" id="p-81" id="p-81" id="p-81"
[0081] The term "effective amount" of an agent as used herein, e.g., a pharmaceutical formulation, refers to an amount effective ,at dosages and for periods of time necessary to, achieve the desired therapeuti cor prophylacti cresult. 82. 82. id="p-82" id="p-82" id="p-82" id="p-82" id="p-82" id="p-82" id="p-82" id="p-82" id="p-82" id="p-82" id="p-82" id="p-82"
[0082] The term "Fc region" as used herein is used to define a C-terminal region of an immunoglobul inheavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment ,a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain. However, the C-terminal lysin e(Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest ,Sth Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991. 83. 83. id="p-83" id="p-83" id="p-83" id="p-83" id="p-83" id="p-83" id="p-83" id="p-83" id="p-83" id="p-83" id="p-83" id="p-83"
[0083] The term "framework" or "ER" as used herein refers to variable domain residues other than hypervariable region (HVR or Hl-3 in the heavy chain and Ll-3 in the light chain) residues The. ER of a variable domain generally consists of four ER domains: ERI, FR2, FR3, and FR4. Accordingly, the HVR and ER sequence sgenerally appear in the following sequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4. 84. 84. id="p-84" id="p-84" id="p-84" id="p-84" id="p-84" id="p-84" id="p-84" id="p-84" id="p-84" id="p-84" id="p-84" id="p-84"
[0084] The term "full length antibody," "intact antibody," or "whol eantibody" refers to an antibody which comprises an antigen-binding variable region (Vh or Vl) as wel las a light chain constant domain (CL) and heavy chain constant domains, CHI, CH2 and CH3. The constant domains may be native sequence constant domains (e.g. human native sequence constant domains) or amino acid sequence variants thereof. Depending on the amino acid 18WO 2020/092155 PCT/US2019/058066 sequence of the constant domain of their heavy chains, full length antibodies can be assigned to different "classes". There are five major classes of full length antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into "subclasses" (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chain constant domains that correspond to the different classes of antibodies are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are wel lknown. 85. 85. id="p-85" id="p-85" id="p-85" id="p-85" id="p-85" id="p-85" id="p-85" id="p-85" id="p-85" id="p-85" id="p-85" id="p-85"
[0085] The terms "host cell" ,"host cel lline," and "host cel lculture" as used herei nare used interchangeably and refe rto cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cell includes "transformants" and "transformed cells," which include the primary transformed cel land progeny derived therefrom without regard to the number of passages Progeny. may not be completely identical in nuclei cacid content to a parent cell but, may contain mutations. Mutant progeny that have the same function or biologica lactivity as screened or selected for in the originall ytransformed cel lare included herein. 86. 86. id="p-86" id="p-86" id="p-86" id="p-86" id="p-86" id="p-86" id="p-86" id="p-86" id="p-86" id="p-86" id="p-86" id="p-86"
[0086] The term "human antibody" as used herein is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human or a human cel lor derived from a non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences. This definition of a human antibody specificall excludesy a humanized antibody comprising non-human antigen-binding residues. 87. 87. id="p-87" id="p-87" id="p-87" id="p-87" id="p-87" id="p-87" id="p-87" id="p-87" id="p-87" id="p-87" id="p-87" id="p-87"
[0087] The term "human consensus framework" as used herein is a framework which represents the most commonly occurring amino acid residues in a selection of human immunoglobul inVl or Vh framework sequence s.Generally, the selection of human immunoglobul inVl or Vh sequence sis from a subgroup of variable domain sequences.
Generally, the subgroup of sequence sis a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest ,Fifth Edition, NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3. In one embodiment ,for the Vl, the subgroup is subgroup kappa I as in Kabat et al., supra. In one embodiment ,for the Vh, the subgroup is subgroup III as in Kabat et al., supra. 88. 88. id="p-88" id="p-88" id="p-88" id="p-88" id="p-88" id="p-88" id="p-88" id="p-88" id="p-88" id="p-88" id="p-88" id="p-88"
[0088] The term "humanized" antibody as used herein refers to a chimeri cantibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody wil lcomprise substantiall ally of at least one, and typicall ytwo, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantiall ally of the FRs correspond to those of a human antibody. A humanize dantibody optionall ymay comprise at 19WO 2020/092155 PCT/US2019/058066 least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization. 89. 89. id="p-89" id="p-89" id="p-89" id="p-89" id="p-89" id="p-89" id="p-89" id="p-89" id="p-89" id="p-89" id="p-89" id="p-89"
[0089] The term "hypervariable region" or "HVR" as used herein refers to each of the regions of an antibody variable domain which are hypervariable in sequence and/or form structurally defined loops ("hypervariable loops"). Generall y,native four-chain antibodies comprise six HVRs; three in the Vh (Hl, H2, H3), and three in the Vl (LI, L2, L3). HVRs generally comprise amino acid residues from the hypervariable loops and/or from the "complementarity determining regions" (CDRs), the latte rbeing of highest sequence variability and/or involved in antigen recognition. Exemplary hypervariable loops occur at amino acid residues 26-32 (LI), 50-52 (L2), 91-96 (L3), 26-32 (Hl), 53-55 (H2), and 96-101 (H3). (Chothia and Lesk, J. Mol. Biol., vol. 196, pp. 901-917 1987) Exemplary CDRs (CDR- LI, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) occur at amino acid residues 24- 34 of LI, 50-56 of L2, 89-97 of L3, 31-35B of Hl, 50-65 of H2, and 95-102 of H3 (Rabat et al., Sequences of Proteins of Immunological Interest ,Sth Ed. Public Health Service, National Institutes of Health, Bethesda, Md. 1991). With the exception of CDR1 in Vh, CDRs generally comprise the amino acid residues that form the hypervariable loops. CDRs also comprise "specificity determining residues," or "SDRs," which are residues that contact antigen. SDRs are contained within regions of the CDRs called abbreviated-CDRs, or a- CDRs. Exemplary a-CDRs (a-CDR-Ll, a-CDR-L2, a-CDR-L3, a-CDR-Hl, a-CDR-H2, and a-CDR-H3) occur at amino acid residues 31-34 of LI, 50-55 of L2, 89-96 of L3, 31-35B of Hl, 50-58 of H2, and 95-102 of H3. (See Almagro and Fransson, Front. Biosci., vol. 13, pp.1619-1633, 2008). Unles sotherwise indicated, HVR residues and other residue sin the variable domain (e.g., ER residues) are numbered herei naccording to Rabat et al., supra. 90. 90. id="p-90" id="p-90" id="p-90" id="p-90" id="p-90" id="p-90" id="p-90" id="p-90" id="p-90" id="p-90" id="p-90" id="p-90"
[0090] The term "immunoconjugate" as used herei nis an antibody or antibody fragment conjugated to one or more heterologous molecule(s), including but not limited to a cytotoxic agent, a chemotherapeuti agent,c a radioactive atom, or a cytostatic agent. 91. 91. id="p-91" id="p-91" id="p-91" id="p-91" id="p-91" id="p-91" id="p-91" id="p-91" id="p-91" id="p-91" id="p-91" id="p-91"
[0091] The term "individual" or "subject" as used herein refers to a mammal. Mammals include, but are not limite dto, domesticated animals (e.g., cows, sheep, cats, dogs, and horses ),primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments the, individual or subject is a human. 92. 92. id="p-92" id="p-92" id="p-92" id="p-92" id="p-92" id="p-92" id="p-92" id="p-92" id="p-92" id="p-92" id="p-92" id="p-92"
[0092] The term "inhibiting cell growth or proliferatio"n as used herei nmeans decreasing a cell 'sgrowth or proliferation by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%, and includes inducing cel ldeath. 20WO 2020/092155 PCT/US2019/058066 93. 93. id="p-93" id="p-93" id="p-93" id="p-93" id="p-93" id="p-93" id="p-93" id="p-93" id="p-93" id="p-93" id="p-93" id="p-93"
[0093] The term "isolated" antibody as used herein is one which has been separated from a component of its natural environment. In some embodiments an, antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoret (e.g.,ic SDS- PAGE, isoelectri focusingc (IEF), capillary electrophoresi ors) chromatographic (e.g., ion exchange or reverse phase High Performance Liquid Chromatography (HPLC)). For review of methods for assessment of antibody purity, see, e.g., Flatman et al., J. Chromatogr. B, vol. 848, pp. 79-87, 2007. 94. 94. id="p-94" id="p-94" id="p-94" id="p-94" id="p-94" id="p-94" id="p-94" id="p-94" id="p-94" id="p-94" id="p-94" id="p-94"
[0094] The term "isolated" nuclei cacid as used herein refers to a nuclei cacid molecule that has been separated from a component of its natural environment .An isolated nucleic acid includes a nuclei cacid molecul containede in cells that ordinarily contain the nuclei cacid molecule, but the nuclei cacid molecul ise present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location. 95. 95. id="p-95" id="p-95" id="p-95" id="p-95" id="p-95" id="p-95" id="p-95" id="p-95" id="p-95" id="p-95" id="p-95" id="p-95"
[0095] The term "isolated nucleic acid encoding an anti-CTLA4 antibody" as used herein refers to one or more nucleic acid molecules encoding antibody heavy and light chains (or fragments thereof), including such nuclei cacid molecule(s in) a singl evector or separate vectors ,and such nuclei cacid molecule(s presen) tat one or more locations in a host cell. 96. 96. id="p-96" id="p-96" id="p-96" id="p-96" id="p-96" id="p-96" id="p-96" id="p-96" id="p-96" id="p-96" id="p-96" id="p-96"
[0096] The term "ligand-independen" tas used herein, as for example applie dto receptor signaling activity, refers to signaling activity that is not dependent on the presence of a ligand. A receptor having ligand-independent kinase activity will not necessarily preclude the binding of ligand to that receptor to produce additional activation of the kinase activity. 97. 97. id="p-97" id="p-97" id="p-97" id="p-97" id="p-97" id="p-97" id="p-97" id="p-97" id="p-97" id="p-97" id="p-97" id="p-97"
[0097] The term "metastasis" as used herein refers to all CTLA4-involving processes that support cancer cells to dispers efrom a primary tumor, penetrate into lymphatic and/or blood vessel circulats, e through the bloodstream, and grow in a distant focus (metastasis) in normal tissues elsewhere in the body. In particular, it refers to cellula eventsr of tumor cells such as proliferation, migration, anchorage independence evasio, n of apoptosis ,or secretion of angiogenic factors, that underli emetastasi sand are stimulate dor mediated by non-catalytic or catalytic activities of CTLA4, preferably including CTLA4 phosphorylation and/or CTLA4-mediated signal transduction. 98. 98. id="p-98" id="p-98" id="p-98" id="p-98" id="p-98" id="p-98" id="p-98" id="p-98" id="p-98" id="p-98" id="p-98" id="p-98"
[0098] The term "microenvironment" as used herein means any portion or region of a tissue or body that has constant or temporal, physical or chemica ldifferences from other regions of the tissue or regions of the body. For tumors, the term "tumor microenvironment" as used herein refers to the environment in which a tumor exists, which is the non-cellula arear within the tumor and the area directly outside the tumorous tissue but does not pertain to the intracellula compar rtment of the cancer cell itself The. tumor and the tumor 21WO 2020/092155 PCT/US2019/058066 microenvironment are closely related and interact constantly. A tumor can change its microenvironmen t,and the microenvironment can affect how a tumor grows and spreads.
Typically, the tumor microenvironment has a low pH in the range of 5.0 to 7.0, or in the range of 5.0 to 6.8, or in the range of 5.8 to 6.8, or in the range of 6.2-6.8. On the other hand, a normal physiological pH is in the range of 7.2-7.8. The tumor microenvironment is also known to have lower concentration of glucose and other nutrients, but higher concentration of lactic acid, in comparison with blood plasma. Furthermore, the tumor microenvironment can have a temperature that is 0.3 to 1 °C higher than the normal physiological temperature. The tumor microenvironment has been discusse din Gillie ets al., "MRI of the Tumor Microenvironment," Journal of Magnetic Resonance Imaging, vol. 16, pp.430-450, 2002, hereby incorporated by reference herein its entirety. The term "non-tumor microenvironment" refers to a microenvironment at a site other than a tumor. 99. 99. id="p-99" id="p-99" id="p-99" id="p-99" id="p-99" id="p-99" id="p-99" id="p-99" id="p-99" id="p-99" id="p-99" id="p-99"
[0099] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies ,i.e., the individual antibodies comprising the population are identical and/or bind the same epitope ,except for possible variant antibodies ,e.g., containing naturall yoccurring mutations or arising during production of a monoclonal antibody preparation, such variants generall beingy presen tin minor amounts .In contrast to polyclonal antibody preparations, which typicall yinclude different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a singl edeterminant on an antigen. Thus, the modifier "monoclona"l indicates the character of the antibody as being obtained from a substantiall homogeneousy population of antibodies ,and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by a variety of techniques, including but not limite dto the hybridoma method, recombinan tDNA methods, phage- display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobul inloci, such methods and other exemplary methods for making monoclonal antibodies being described herein. 100. 100. id="p-100" id="p-100" id="p-100" id="p-100" id="p-100" id="p-100" id="p-100" id="p-100" id="p-100" id="p-100" id="p-100" id="p-100"
[0100] The term "naked antibody" as used herein refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or radiolabel. The naked antibody may be present in a pharmaceutical formulation. 101. 101. id="p-101" id="p-101" id="p-101" id="p-101" id="p-101" id="p-101" id="p-101" id="p-101" id="p-101" id="p-101" id="p-101" id="p-101"
[0101] The term "native antibodies" as used herei nrefers to naturall yoccurring immunoglobul inmolecules with varying structures. For example, native IgG antibodies are heterotetrameric glycoprotein sof about 150,000 daltons, composed of two identical light 22WO 2020/092155 PCT/US2019/058066 chains and two identical heavy chains that are disulfide-bonded. From N- to C-terminus, each heavy chain has a variable region (Vh), also called a variable heavy domain or a heavy chain variable domain, followed by three constant domains (CHI, CH2, and CH3). Similarl y,from N- to C-terminus ,each light chain has a variable region (Vl), also called a variable light domain or a light chain variable domain, followed by a constant light (Cl) domain. The light chain of an antibody may be assigned to one of two types ,called kappa (k) and lambda (X), based on the amino acid sequence of its constant domain. 102. 102. id="p-102" id="p-102" id="p-102" id="p-102" id="p-102" id="p-102" id="p-102" id="p-102" id="p-102" id="p-102" id="p-102" id="p-102"
[0102] The term "package insert" as used herei nis used to refer to instructions customaril y included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy ,contraindications and/or warnings concerning the use of such therapeutic products. 103. 103. id="p-103" id="p-103" id="p-103" id="p-103" id="p-103" id="p-103" id="p-103" id="p-103" id="p-103" id="p-103" id="p-103" id="p-103"
[0103] The term "percent (%) amino acid sequence identity" with respec tto a reference polypeptide sequence as used herei nis defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly availabl ecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software .Those skill edin the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared .For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly availabl efrom Genentech, Inc., South San Francisco, Calif., or may be compiled from the source code. The ALIGN-2 program should be compiled for use on a UNIX operating system, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary. 104. 104. id="p-104" id="p-104" id="p-104" id="p-104" id="p-104" id="p-104" id="p-104" id="p-104" id="p-104" id="p-104" id="p-104" id="p-104"
[0104] In situations where ALIGN-2 is employed for amino acid sequence comparisons, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternativel bey phrased as a given amino acid sequence A 23WO 2020/092155 PCT/US2019/058066 that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculate asd follows: 100 times the fraction X/Y where X is the number of amino acid residue sscored as identical matches by the sequence alignment program ALIGN-2 in that program's alignmen oft A and B, and where Y is the total number of amino acid residues in B. It wil lbe appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B wil lnot equal the % amino acid sequence identity of B to A.
Unless specificall staty ed otherwise, all % amino acid sequence identity values used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program. 105. 105. id="p-105" id="p-105" id="p-105" id="p-105" id="p-105" id="p-105" id="p-105" id="p-105" id="p-105" id="p-105" id="p-105" id="p-105"
[0105] The term "pharmaceutical formulation" as used herei nrefers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective ,and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered. 106. 106. id="p-106" id="p-106" id="p-106" id="p-106" id="p-106" id="p-106" id="p-106" id="p-106" id="p-106" id="p-106" id="p-106" id="p-106"
[0106] The term "pharmaceutical lyacceptable carrier" as used herein refers to an ingredient in a pharmaceutical formulation, other than an active ingredient ,which is nontoxic to a subject., A pharmaceutical lyacceptable carrier includes but, is not limite dto, a buffer, excipient, stabilize r,or preservative. 107. 107. id="p-107" id="p-107" id="p-107" id="p-107" id="p-107" id="p-107" id="p-107" id="p-107" id="p-107" id="p-107" id="p-107" id="p-107"
[0107] The terms "purified" and "isolated" used herei nrefe rto an antibody according to the invention or to a nucleotide sequence, that the indicated molecul ise presen tin the substantial absence of other biologica lmacromolecules of the same type. The term "purified" as used herein preferably means at leas 75%t by weight ,more preferably at leas 85%t by weight, more preferably still at leas 95%t by weight ,and most preferably at least 98% by weight ,of biological macromolecules of the same type are present. An "isolated" nucleic acid molecul e which encodes a particular polypeptide refers to a nucleic acid molecul whice h is substantiall freey of other nucleic acid molecules that do not encode the polypeptide; however, the molecul maye include some additional bases or moieties which do not deleteriousl affecy t the basic characteristics of the composition. 108. 108. id="p-108" id="p-108" id="p-108" id="p-108" id="p-108" id="p-108" id="p-108" id="p-108" id="p-108" id="p-108" id="p-108" id="p-108"
[0108] The term "recombinant antibody" as used herei nrefers to an antibody (e.g. a chimeric ,humanized, or human antibody or antigen-binding fragment thereof) that is expressed by a recombinant host cell comprising nucleic acid encoding the antibody.
Examples of "host cells" for producing recombinant antibodies include: (1) mammalian cell s, for example, Chinese Hamster Ovary (CHO), COS, myeloma cells (including Y0 and NS0 24WO 2020/092155 PCT/US2019/058066 cells), baby hamster kidney (BHK), Hel aand Vero cells; (2) insec tcells, for example, sf9, sf21 and Tn5; (3) plant cells, for exampl eplants belongin gto the genus Nicotiana (e.g.
Nicotiana tabacumY, (4) yeast cells, for example, those belonging to the genus Saccharomyces (e.g. Saccharomyces cerevisiae) or the genus Aspergillus (e.g. Aspergillus nigerY (5) bacterial cells, for exampl eEscherichia, coll cell ors Bacillus subtilis cell s,etc. 109. 109. id="p-109" id="p-109" id="p-109" id="p-109" id="p-109" id="p-109" id="p-109" id="p-109" id="p-109" id="p-109" id="p-109" id="p-109"
[0109] The term "CTLA4" as used herein, refers to an immune checkpoint that has the amino acid sequence as described in U.S. Patent Nos. 5,434,131, 5,844,095, and 5,851,795, or any portion or derivative thereof that, recognizes and binds a B7 or interferes with a B7 so that it blocks binding to CD28 and/or CTLA4 (e.g., endogenous CD28 and/or CTLA4). In particular embodiments, the extracellula domainr of wild type CTLA4 begins with methionine at position +1 and ends at aspartic acid at position +124, or the extracellula domainr of wild type CTLA4 begins with alanine at position -1 and ends at aspartic acid at position +124.
Wild type CTLA4 is a cel lsurface protein, having an N-terminal extracellula domair n, a transmembrane domain, and a C-terminal cytoplasmi cdomain. The extracellula domainr binds to target molecules, such as a B7 molecule. In a cell the, naturally occurring, wild type CTLA4 protein is translated as an immature polypeptide, which includes a signal peptide at the N-terminal end. The immature polypeptide undergoes post-translationa processil ng, which includes cleavage and removal of the signal peptide to generate a CTLA4 cleavage product having a newly generated N-terminal end that differs from the N-terminal end in the immature form. One skilled in the art wil lappreciate that additional post-translationa l processing may occur, which removes one or more of the amino acids from the newly generated N-terminal end of the CTLA4 cleavage product. Alternatively, the signal peptide may not be removed completel generaty, ing molecules that begin before the common starting amino acid methionine. Thus, the mature CTLA4 protein may start at methionine at position +1 or alanine at position -1. The mature form of the CTLA4 molecule includes the extracellula domainr or any portion thereof. 110. 110. id="p-110" id="p-110" id="p-110" id="p-110" id="p-110" id="p-110" id="p-110" id="p-110" id="p-110" id="p-110" id="p-110" id="p-110"
[0110] The term "therapeutically effective amount" of the antibody of the invention is meant a sufficient amount of the antibody to treat said cancer, at a reasonable benefit/risk ratio applicable to any medical treatment. It wil lbe understood, however, that the total daily usage of the antibodies and compositions of the present invention wil lbe decided by the attending physician within the scope of sound medical judgment .The specific therapeutically effective dose level for any particular patient wil ldepend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific antibody employe d;the specific composition employed, the age, body weight, genera lhealth, sex and 25WO 2020/092155 PCT/US2019/058066 diet of the patient; the time of administration, route of administration, and rate of excretion of the specific antibody employe d;the duration of the treatment ;drugs used in combination or coincidental with the specific antibody employe d;and like factors wel lknown in the medical arts. For example, it is wel lknown within the skil ofl the art to start doses of the compound at level lowers than those required to achieve the desired therapeuti ceffect and to gradually increase the dosage until the desired effect is achieved. 111. 111. id="p-111" id="p-111" id="p-111" id="p-111" id="p-111" id="p-111" id="p-111" id="p-111" id="p-111" id="p-111" id="p-111" id="p-111"
[0111] The term "singl echain Fv" ("scFv") as used herei nis a covalently linked Vh::Vl heterodime whicr h is usually expressed from a gene fusion including Vh and Vl encoding genes linked by a peptide-encoding linker. "dsFv" is a Vh::Vl heterodimer stabilise byd a disulfide bond. Divalent and multivalent antibody fragments can form eithe rspontaneousl y by association of monovalent scFvs, or can be generated by coupling monovalen scFvst by a peptide linker, such as divalent sc(Fv)2. 112. 112. id="p-112" id="p-112" id="p-112" id="p-112" id="p-112" id="p-112" id="p-112" id="p-112" id="p-112" id="p-112" id="p-112" id="p-112"
[0112] The term "treatment," "treat," or "treating" as used herei nrefers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed eithe rfor prophylaxis or during the course of clinical pathology. Desirabl e effects of treatment include ,but are not limite dto, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the diseas estate, and remission or improved prognosis. In some embodiments antibodie, s of the invention are used to dela ydevelopme nt of a diseas eor to slow the progression of a disease. 113. 113. id="p-113" id="p-113" id="p-113" id="p-113" id="p-113" id="p-113" id="p-113" id="p-113" id="p-113" id="p-113" id="p-113" id="p-113"
[0113] The term "tumor" as used herein refers to all neoplasti ccel lgrowth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues The . terms "cancer," "cancerous," "cel lproliferative disorder," "proliferative disorder" and "tumor" are not mutuall yexclusive as referred to herein. 114. 114. id="p-114" id="p-114" id="p-114" id="p-114" id="p-114" id="p-114" id="p-114" id="p-114" id="p-114" id="p-114" id="p-114" id="p-114"
[0114] The term "variable region" or "variable domain" as used herein refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable domains of the heavy chain and light chain (Vh and Vl, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindt et al.
Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007).) A singl eVh or Vl domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen may be isolated using a Vh or Vl domain from an antibody that binds the antigen to screen a library of complementary Vl or Vh domains, respectively. See, 26WO 2020/092155 PCT/US2019/058066 e.g., Portolano et al., J. Immunol., vol. 150, pp. 880-887, 1993; Clarkson et al., Nature, vol. 352, pp. 624-628, 1991. 115. 115. id="p-115" id="p-115" id="p-115" id="p-115" id="p-115" id="p-115" id="p-115" id="p-115" id="p-115" id="p-115" id="p-115" id="p-115"
[0115] The term "vector" as used herein refers to a nucleic acid molecul capablee of propagating another nucleic acid to which it is linked. The term includes the vector as a self - replicating nucleic acid structure as wel las the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nuclei cacids to which they are operativel ylinked. Such vectors are referred to herein as "expression vectors." DETAILED DESCRIPTION 116. 116. id="p-116" id="p-116" id="p-116" id="p-116" id="p-116" id="p-116" id="p-116" id="p-116" id="p-116" id="p-116" id="p-116" id="p-116"
[0116] For illustrative purposes, the principles of the presen tinvention are described by referencing various exemplary embodiments. Although certain embodiment sof the invention are specificall descy ribed herein, one of ordinary skil inl the art wil lreadily recognize that the same principle sare equally applicable to, and can be employed in, other systems and methods. Before explaining the disclose embodd iments of the present invention in detail, it is to be understood that the invention is not limited in its application to the details of any particular embodiment shown .Additionally, the terminology used herein is for the purpose of description and not for limitation. Furthermore, although certain methods are described with reference to steps that are presented herein in a certain order, in many instances, thes esteps can be performed in any order as may be appreciated by one skilled in the art; the novel method is therefore not limite dto the particular arrangement of steps disclose hereid n. 117. 117. id="p-117" id="p-117" id="p-117" id="p-117" id="p-117" id="p-117" id="p-117" id="p-117" id="p-117" id="p-117" id="p-117" id="p-117"
[0117] It must be noted that as used herei nand in the appended claims the, singular forms "a", "an", and "the" include plural references unless the context clearl dicty ates otherwise.
Furthermore, the terms "a" (or "an"), "one or more", and "at least one" can be used interchangeably herein. The terms "comprising", "including", "having" and "constructed from" can also be used interchangeably. 118. 118. id="p-118" id="p-118" id="p-118" id="p-118" id="p-118" id="p-118" id="p-118" id="p-118" id="p-118" id="p-118" id="p-118" id="p-118"
[0118] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, percent, ratio, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about," whether or not the term "about" is present. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present disclosure. At the very least, and not as an attempt to limi tthe application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be 27WO 2020/092155 PCT/US2019/058066 construed in light of the number of reported significant digits and by applying ordinary rounding techniques Notw. ithstanding that the numerical ranges and parameters setting forth the broad scope of the disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. 119. 119. id="p-119" id="p-119" id="p-119" id="p-119" id="p-119" id="p-119" id="p-119" id="p-119" id="p-119" id="p-119" id="p-119" id="p-119"
[0119] It is to be understood that each component, compound, substituent, or parameter disclosed herei nis to be interpreted as being disclose ford use alone or in combination with one or more of each and every other component, compound, substituent ,or paramete r disclosed herein. 120. 120. id="p-120" id="p-120" id="p-120" id="p-120" id="p-120" id="p-120" id="p-120" id="p-120" id="p-120" id="p-120" id="p-120" id="p-120"
[0120] It is also to be understood that each amount/value or range of amounts/values for each component, compound, substituent, or parameter disclosed herein is to be interpreted as also being disclosed in combination with each amount/value or range of amounts/value discloseds for any other component(s), compounds(s), substituent(s), or parameter(s) disclose hereind and that any combination of amounts/values or ranges of amounts/values for two or more component(s), compounds(s) ,substituent(s), or parameters disclose hereind are thus also disclosed in combination with each other for the purposes of this description. 121. 121. id="p-121" id="p-121" id="p-121" id="p-121" id="p-121" id="p-121" id="p-121" id="p-121" id="p-121" id="p-121" id="p-121" id="p-121"
[0121] It is further understood that each lower limi tof each range disclosed herei nis to be interpreted as disclosed in combination with each upper limi tof each range disclosed herei n for the same component ,compounds, substituent ,or parameter. Thus, a disclosure of two ranges is to be interpreted as a disclosure of four ranges derived by combining each lower limi tof each range with each upper limit of each range. A disclosure of three ranges is to be interpreted as a disclosure of nine ranges derived by combining each lower limi tof each range with each upper limi tof each range, etc. Furthermore, specific amounts/values of a component, compound, substituent, or parameter disclosed in the description or an exampl eis to be interpreted as a disclosure of either a lower or an upper limi tof a range and thus can be combined with any other lower or upper limi tof a range or specific amount/value for the same component, compound, substituent ,or parameter disclosed elsewhere in the application to form a range for that component, compound, substituent ,or parameter.
A. Anti-CTLA4 Antibodies 122. 122. id="p-122" id="p-122" id="p-122" id="p-122" id="p-122" id="p-122" id="p-122" id="p-122" id="p-122" id="p-122" id="p-122" id="p-122"
[0122] In one aspect, the presen tinvention provides an isolated heavy chain variable region polypeptide that specificall bindsy to human CTLA4 protein. The isolated heavy chain 28WO 2020/092155 PCT/US2019/058066 variable region polypeptide comprises three complementarity determining regions having the Hl, H2, and H3, wherein: the Hl sequence is GFTFSHYTMH (SEQ ID NO: 1); the H2 sequence is FIXYX2GNXKX4X5AX6SX7KG (SEQ ID NO: 2); and the H3 sequence is TGWLGPFDXs (SEQ ID NO: 3), wherein X! is S or D; X2 is D, H or I, X3 is N or Y; X4 is Y or I; X5 is Y or E; X6 is D or K; X? is V or M; and X8 is Y or I. 123. 123. id="p-123" id="p-123" id="p-123" id="p-123" id="p-123" id="p-123" id="p-123" id="p-123" id="p-123" id="p-123" id="p-123" id="p-123"
[0123] The alignment sof exemplary isolated heavy chain variable regions of the present invention are shown in FIG. 1, where the complementarit detey rmining regions Hl, H2, and H3 are boxed. 124. 124. id="p-124" id="p-124" id="p-124" id="p-124" id="p-124" id="p-124" id="p-124" id="p-124" id="p-124" id="p-124" id="p-124" id="p-124"
[0124] In another aspect, the present invention provides an isolated light chain variable region polypeptide that specifically binds to human CTLA4 protein. The isoloated light chain variable region polypeptide comprises three complementarity determining regions having the sequences LI, L2, and L3, wherein: the LI sequence is RX9SQX0X1GSSYLA (SEQ ID NO: 4); the L2 sequence is GAFSRATGX12 (SEQ ID NO: 5); and the L3 sequence is QQDGSSPWT (SEQ ID NO: 6), wherein Xg is A or I; X10 is Y, S or H; Xu is V or G; X!2 is V or I. 125. 125. id="p-125" id="p-125" id="p-125" id="p-125" id="p-125" id="p-125" id="p-125" id="p-125" id="p-125" id="p-125" id="p-125" id="p-125"
[0125] The alignment sof exemplary isolated light chain variable regions of the present invention are shown in FIG. 2, where the complementarit detey rmining regions LI, L2, and L3 are boxed. 126. 126. id="p-126" id="p-126" id="p-126" id="p-126" id="p-126" id="p-126" id="p-126" id="p-126" id="p-126" id="p-126" id="p-126" id="p-126"
[0126] The isolated heavy chain variable region polypeptides and the isolated light chain variable region polypeptides of the presen tinvention were each obtained from a parent antibody using a method disclosed in U.S. Patent No. 8,709,755. This method of generating The isolated heavy chain variable region polypeptides and the isolated light chain variable region polypeptides, as wel las the method of generating antibodies and antibody fragments disclosed in U.S. Patent No. 8,709,755 are hereby incorporated by reference herein. 127. 127. id="p-127" id="p-127" id="p-127" id="p-127" id="p-127" id="p-127" id="p-127" id="p-127" id="p-127" id="p-127" id="p-127" id="p-127"
[0127] In another aspect, the present invention includes the heavy chain variable regions shown in FIG. 1 and the light chain variable regions shown in FIG. 2. The amino acid sequences of the 16 heavy chain variable regions of Fig. 1 are set forth in SEQ ID NOS: 8, , 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38. The amino acid sequences of the 16 light chain variable regions of Fig. 1 are set forth in SEQ ID NOS: 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37. Antibodies and antibody fragments including these heavy chain variable regions and light chain variable regions can specifically bind to human CTLA4. 29WO 2020/092155 PCT/US2019/058066 Antibodies or antibody fragments comprising a combination of one of these heavy chain variable regions and one of these light chain variable regions have been found to have higher binding affinity to CTLA4 at a pH in the tumor microenvironment (e.g. pH 6.0-6.2) than at a pH in a non-tumor microenvironment (e.g. pH 7.4). As a resul t,the anti-CTLA4 antibodies or antibody fragments have a higher binding affinity to CTLA4 in a tumor microenvironment in comparison with their binding affinity to CTLA4 in a normal tissue microenvironment. 128. 128. id="p-128" id="p-128" id="p-128" id="p-128" id="p-128" id="p-128" id="p-128" id="p-128" id="p-128" id="p-128" id="p-128" id="p-128"
[0128] Anti-CTLA4 antibodies or antibody fragments of the present invention thus have reduced side-effects due to their reduced binding affinity to CTLA4 in the normal tissue microenvironmen t,as wel las comparable efficacy, in comparison with monoclonal anti- CTLA4 antibodies known in the art. These features permit use of a higher dosage of these anti-CTLA4 antibodies or antibody fragments to be delivered to a patient thus providing a more effective therapeuti coption. 129. 129. id="p-129" id="p-129" id="p-129" id="p-129" id="p-129" id="p-129" id="p-129" id="p-129" id="p-129" id="p-129" id="p-129" id="p-129"
[0129] Though the present invention includes the heavy chain variable regions and light chain variable regions presented in FIGS. 1-2 and those having amino acid sequences of SEQ ID NOS: 7-38, the present invention also includes variants thereof that can specificall bindy to human CTLA4. In some embodiments, thes evariants have different H2, H3,11 and 12 sequences. In other embodiments, the amino acid sequence of the heavy chain variable regions and light chains variable regions outside of the complementarity determining regions may be mutated in accordance with the principles of substitution, insertion and deletion as discussed in this application. In still further embodiments, the constant regions may be modified to provide variants. 130. 130. id="p-130" id="p-130" id="p-130" id="p-130" id="p-130" id="p-130" id="p-130" id="p-130" id="p-130" id="p-130" id="p-130" id="p-130"
[0130] In deriving these variants, one is guided by the process as described herein. The variants of the heavy chain variable regions and light chain variable regions may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the heavy chain variable regions and light chain variable regions, or by peptide synthesi s.Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of residues within the amino acid sequences of the heavy chain variable regions and light chain variable regions. Any combination of deletion, insertion, and substitution can be made to arrive at the antibodies or antibody fragments of the present invention, provided that they posses sthe desired characteristics, e.g., antigen-binding to human CTLA4 and/or conditional activity. 30WO 2020/092155 PCT/US2019/058066 Substitution, Insertion, and Deletion Variants 131. 131. id="p-131" id="p-131" id="p-131" id="p-131" id="p-131" id="p-131" id="p-131" id="p-131" id="p-131" id="p-131" id="p-131" id="p-131"
[0131] In certain embodiments, antibody or antibody fragment variants having one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesi sinclude the CDRs and framework regions (FRs). Conservative substitutions are shown in Table 1 under the heading of "conservative substitutions." More substantial changes are provided in Table 1 under the heading of "exemplary substitutions," and as further described below in reference to amino acid side chain classes. Amino acid substitutions may be introduced into an antibody or antibody fragment of interest and the products screened for a desired activity, e.g., retained/improved antigen binding, or decreased immunogenicity. 31WO 2020/092155 PCT/US2019/058066 Table 1: Amino acid substitutions □ □ ח Original Exemplary Preferred n Residue Substitutions Substitutions Ala (A) Vai; Leu; He Vai Arg(R) Lys; Gin; Asn Lys Asn (N) Gin; His; Asp, Lys; Arg Gin Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gin (Q) Asn; Glu Asn Glu (E) Asp; Gin Asp Gly (G) Ala Ala His (H) Asn; Gin; Lys; Arg Arg Leu; Vai; Met; Ala; Phe; NorleucineLeu He (I) Leu (L) Norleucine; He; Vai; Met; Ala; Phe He Lys (K) Arg; Gin; Asn Arg Met (M) Leu; Phe; He Leu Phe (F) Trp; Leu; Vai; He; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr(T) Vai; Ser Ser Trp (W) Tyr; Phe Tyr Trp; Phe; Thr; Ser Phe Tyr (Y) fl!Va i(V) He; Leu; Met; Phe; Ala; Norleucine Leu 132. 132. id="p-132" id="p-132" id="p-132" id="p-132" id="p-132" id="p-132" id="p-132" id="p-132" id="p-132" id="p-132" id="p-132" id="p-132"
[0132] Amino acids may be grouped according to common side-chain properties: (1) hydrophobic :Norleucine, Met, Ala, Vai, Leu, lie; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. 133. 133. id="p-133" id="p-133" id="p-133" id="p-133" id="p-133" id="p-133" id="p-133" id="p-133" id="p-133" id="p-133" id="p-133" id="p-133"
[0133] Non-conservative substitutions will entail exchanging a member of one of these classes for another class. 134. 134. id="p-134" id="p-134" id="p-134" id="p-134" id="p-134" id="p-134" id="p-134" id="p-134" id="p-134" id="p-134" id="p-134" id="p-134"
[0134] One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g. a humanized or human antibody). Generally, the resulting variant(s) selected for further study wil lhave modifications (e.g., improvements) in certain biologica lproperties (e.g., increased affinity, reduced immunogenicity) relative to the 32WO 2020/092155 PCT/US2019/058066 parent antibody and/or wil lhave substantiall retay ined certain biologica lproperties of the parent antibody. An exemplary substitutional variant is an affinity matured antibody, which may be convenientl ygenerated, e.g., using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more CDR residue sare mutated and the variant antibodies displayed on phage and screened for a particular biological activity (e.g. binding affinity). 135. 135. id="p-135" id="p-135" id="p-135" id="p-135" id="p-135" id="p-135" id="p-135" id="p-135" id="p-135" id="p-135" id="p-135" id="p-135"
[0135] Alterations (e.g., substitutions) may be made in CDRs, e.g., to improve antibody affinity. Such alterations may be made in CDR "hotspots", i.e., residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol., vol. 207, pp. 179-196, 2008), and/or SDRs (a-CDRs), with the resulting variant VH or VL being tested for binding affinity. Affinity maturation by constructing and reselecting from secondary libraries has been described, e.g., in Hoogenboom et al. in Methods in Molecular Biology, vol. 178, pp. 1-37, 2001). In some embodiments of affinity maturation, diversity is introduced into the variable genes chosen for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another method to introduce diversity involves CDR-directed approaches, in which several CDR residues (e.g., 4-6 residues at a time) are randomized. CDR residues involved in antigen binding may be specifically identified, e.g., using alanine scanning mutagenesi sor modeling. CDR-H3 and CDR-L3 in particular are often targeted. 136. 136. id="p-136" id="p-136" id="p-136" id="p-136" id="p-136" id="p-136" id="p-136" id="p-136" id="p-136" id="p-136" id="p-136" id="p-136"
[0136] In certain embodiments, substitutions, insertions, or deletions may occur within one or more HVRs so long as such alterations do not substantially reduce the ability of the antibody or antibody fragment to bind antigen. For example, conservative alterations (e.g., conservative substitutions as provided herein) that do not substantiall reducey binding affinity may be made in CDRs. Such alterations may be outside of CDR "hotspots" or SDRs. In certain embodiments of the variant Vh and Vl sequences provided above, each CDR eithe ris unaltered, or contains no more than one, two or three amino acid substitutions. 137. 137. id="p-137" id="p-137" id="p-137" id="p-137" id="p-137" id="p-137" id="p-137" id="p-137" id="p-137" id="p-137" id="p-137" id="p-137"
[0137] A useful method for identification of residues or regions of an antibody that may be targeted for mutagenesi sis called "alanine scanning mutagenesi"s as described by Cunningham and Wells Science, , vol. 244, pp. 1081-1085, 1989. In this method, a residue or group of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) are identified and replaced by a neutral or negatively charged amino acid (e.g., alanine or polyalanine to) determine whether the interaction of the antibody or antibody fragment with 33WO 2020/092155 PCT/US2019/058066 antigen is affected. Further substitutions may be introduced at the amino acid locations demonstrating functional sensitivity to the initial substitutions. Alternatively, or additionally, a crystal structure of an antigen-antibody comple xto identify contact points between the antibody or antibody fragment and antigen. Such contact residues and neighboring residues may be targeted or eliminated as candidates for substitution. Variants may be screened to determine whether they contain the desired properties. 138. 138. id="p-138" id="p-138" id="p-138" id="p-138" id="p-138" id="p-138" id="p-138" id="p-138" id="p-138" id="p-138" id="p-138" id="p-138"
[0138] Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues as, well as intrasequenc einsertions of singl eor multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue. Othe r insertional variants of the antibody include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g. for ADEPT) or a polypeptide which increases the serum half-life of the antibody. 139. 139. id="p-139" id="p-139" id="p-139" id="p-139" id="p-139" id="p-139" id="p-139" id="p-139" id="p-139" id="p-139" id="p-139" id="p-139"
[0139] Amino acid sequence modification(s) of the antibodies described herei nare contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody. It is known that when a humanized antibody is produced by simpl ygrafting only CDRs in Vh and Vl of an antibody derived from a non- human animal in FRs of the Vh and Vl of a human antibody, the antigen binding activity is reduced in comparison with that of the original antibody derived from a non-human animal. It is considered that several amino acid residue sof the Vh and Vl of the non-human antibody, not only in CDRs but also in FRs, are directly or indirectly associated with the antigen binding activity. Hence ,substitution of these amino acid residues with different amino acid residues derived from FRs of the Vh and Vl of the human antibody would reduce of the binding activity. In order to resolve the problem, in antibodies grafted with human CDR, attempts have to be made to identify, among amino acid sequences of the FR of the Vh and Vl of human antibodies, an amino acid residue which is directly associated with binding to the antibody, or which interacts with an amino acid residue of CDR, or which maintains the three-dimensional structure of the antibody and which is directly associated with binding to the antigen. The reduced antigen binding activity could be increased by replacing the identified amino acids with amino acid residues of the original antibody derived from a non- human animal. 140. 140. id="p-140" id="p-140" id="p-140" id="p-140" id="p-140" id="p-140" id="p-140" id="p-140" id="p-140" id="p-140" id="p-140" id="p-140"
[0140] Modifications and changes may be made in the structure of the antibodies of the present invention, and in the DNA sequence sencoding them, and still obtain a functional molecul thate encodes an antibody with desirable characteristics. 34WO 2020/092155 PCT/US2019/058066 141. 141. id="p-141" id="p-141" id="p-141" id="p-141" id="p-141" id="p-141" id="p-141" id="p-141" id="p-141" id="p-141" id="p-141" id="p-141"
[0141] In making the changes in the amino sequences the, hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art. It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules , for example, enzymes, substrates recept, ors, DNA, antibodies ,antigens, and the like. Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalani ne(+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (0.7־); serine (0.8־); tryptophane (0.9־); tyrosine (1.3־); proline (1.6־); histidine (3.2־); glutamate (3.5־); glutamine (3.5־); aspartate (3.5־); asparagine (3.5־); lysine (3.9־); and arginine (4.5־). 142. 142. id="p-142" id="p-142" id="p-142" id="p-142" id="p-142" id="p-142" id="p-142" id="p-142" id="p-142" id="p-142" id="p-142" id="p-142"
[0142] A further object of the presen tinvention also encompasses function-conservative variants of the antibodies of the present invention. 143. 143. id="p-143" id="p-143" id="p-143" id="p-143" id="p-143" id="p-143" id="p-143" id="p-143" id="p-143" id="p-143" id="p-143" id="p-143"
[0143] "Function-conservative variants" are those in which a given amino acid residue in a protein or enzyme has been changed without altering the overal lconformation and function of the polypeptide, including, but not limite dto, replaceme ntof an amino acid with one having similar properties (such as, for example, polarity, hydrogen bonding potential ,acidic, basic, hydrophobic, aromatic, and the like). Amino acids other than those indicated as conserved may differ in a protein so that the percent protein or amino acid sequence similarit ybetween any two proteins of similar function may vary and may be, for example, from 70% to 99% as determine daccording to an alignment scheme such as by the Cluster Method, wherei nsimilarity is based on the MEGALIGN algorithm A. "function-conservative variant" also includes a polypeptide which has at least 60% amino acid identity as determined by BLAST or FASTA algorithms, preferably at least 75%, more preferably at least 85%, still preferably at least 90%, and even more preferabl yat least 95%, and which has the same or substantiall simy ilar properties or functions as the native or parent protein to which it is compared. 144. 144. id="p-144" id="p-144" id="p-144" id="p-144" id="p-144" id="p-144" id="p-144" id="p-144" id="p-144" id="p-144" id="p-144" id="p-144"
[0144] Two amino acid sequences are "substantiall homologousy " or "substantiall simy ilar" when greater than 80%, preferably greater than 85%, preferably greater than 90% of the amino acids are identical, or greater than about 90%, preferably greater than 95%, are similar (functionally identical )over the whole length of the shorter sequence. Preferably, the similar or homologous sequence sare identified by alignmen usit ng, for example, the GCG (Genetics 35WO 2020/092155 PCT/US2019/058066 Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wis.) pileup program, or any of sequence comparison algorithms such as BLAST, PASTA, etc. 145. 145. id="p-145" id="p-145" id="p-145" id="p-145" id="p-145" id="p-145" id="p-145" id="p-145" id="p-145" id="p-145" id="p-145" id="p-145"
[0145] For example, certain amino acids may be substituted by othe ramino acids in a protein structure without appreciable los sof activity. Since the interactive capacity and nature of a protein define the protein's biological functional activity, certain amino acid substitutions can be made in a protein sequence, and, of course, in its DNA encoding sequence, while nevertheles obtas ining a protein with like properties .It is thus contemplate thatd various changes may be made in the sequences of the antibodies or antibody fragments of the invention, or corresponding DNA sequences which encode said antibodies or antibody fragments, without appreciable los sof their biologica lactivity. 146. 146. id="p-146" id="p-146" id="p-146" id="p-146" id="p-146" id="p-146" id="p-146" id="p-146" id="p-146" id="p-146" id="p-146" id="p-146"
[0146] It is known in the art that certain amino acids may be substituted by other amino acids having a similar hydropathic index or score and still resul int a protein with similar biological activity, i.e. still obtain a biologica lfunctionall yequivalent protein. 147. 147. id="p-147" id="p-147" id="p-147" id="p-147" id="p-147" id="p-147" id="p-147" id="p-147" id="p-147" id="p-147" id="p-147" id="p-147"
[0147] As outlined above, amino acid substitutions are generally therefore based on the relative similarit yof the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size ,and the like. Exemplary substitutions which take various of the foregoing characteristics into consideration are wel lknown to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine ;and valine ,leucine and isoleucine.
Glycosylation Variants 148. 148. id="p-148" id="p-148" id="p-148" id="p-148" id="p-148" id="p-148" id="p-148" id="p-148" id="p-148" id="p-148" id="p-148" id="p-148"
[0148] In certain embodiments, an antibody provided herein is altered to increase or decrease the extent to which the antibody is glycosylate d.Addition or deletion of glycosylation sites to an antibody may be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites is created or removed. 149. 149. id="p-149" id="p-149" id="p-149" id="p-149" id="p-149" id="p-149" id="p-149" id="p-149" id="p-149" id="p-149" id="p-149" id="p-149"
[0149] Where the antibody comprises an Fc region, the carbohydrate attached thereto may be altered. Native antibodies produced by mammalian cell typics all ycomprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al. TIBTECH, vol. 15, pp. 26-32, 1997. The oligosaccharide may include various carbohydrates, e.g., mannose ,N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as wel las a fucose attached to a GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharide in an antibody of the invention may be made in order to create antibody variants with certain improved properties. 36WO 2020/092155 PCT/US2019/058066 150. 150. id="p-150" id="p-150" id="p-150" id="p-150" id="p-150" id="p-150" id="p-150" id="p-150" id="p-150" id="p-150" id="p-150" id="p-150"
[0150] In one embodiment ,antibody variants are provided having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region. For example, the amount of fucose in such antibody may be from 1% to 80%, from 1% to 65%, from 5% to 65% or from % to 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn 297 (e.g. complex, hybrid and high mannose structures )as measured by MALDI-TOF mass spectrometry as, described in WO 2008/077546, for example. Asn297 refers to the asparagine residue located at about position 297 in the Fc region (Eu numbering of Fc region residues); however, Asn297 may also be located about ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. See, e.g., US Patent Publication Nos. US 2003/0157108 (Presta, L.); US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications relate dto "defucosylated" or "fucose- deficient" antibody variants include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; WO2005/053742; WO2002/031140; Okazaki et al. J.
Mol. Biol., vol. 336, pp. 1239-1249, 2004; Yamane-Ohnuki et al. Biotech. Bioeng., vol. 87, pp. 614-622, 2004. Examples of cell line scapable of producing defucosylated antibodies include Lee 13 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem.
Biophys., vol. 249, pp. 533-545, 1986; US Pat Appl No US 2003/0157108 A; and WO 2004/056312 Al, especial lyat Example 11), and knockout cell lines, such as alpha-1,6- fucosyltransferase gene ,FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng., vol. 87, pp. 614-622, 2004; Kanda, Y. et al., Biotechnol. Bioeng., vol. 94, pp. 680-688, 2006; and WO2003/085107). 151. 151. id="p-151" id="p-151" id="p-151" id="p-151" id="p-151" id="p-151" id="p-151" id="p-151" id="p-151" id="p-151" id="p-151" id="p-151"
[0151] Antibody variants are further provided with bisected oligosaccharides, e.g., in which a biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc.
Such antibody variants may have reduced fucosylation and/or improved ADCC function.
Examples of such antibody variants are described, e.g., in WO 2003/011878; U.S. Pat. No. 6,602,684; and US 2005/0123546. Antibody variants with at least one galactos eresidue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, e.g., in WO 1997/30087; WO 1998/58964; and WO 1999/22764. 37WO 2020/092155 PCT/US2019/058066 Fc Region Variants 152. 152. id="p-152" id="p-152" id="p-152" id="p-152" id="p-152" id="p-152" id="p-152" id="p-152" id="p-152" id="p-152" id="p-152" id="p-152"
[0152] In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant.
The Fc region variant may comprise a human Fc region sequence (e.g., a human IgGl ,IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g. a substitution) at one or more amino acid positions. 153. 153. id="p-153" id="p-153" id="p-153" id="p-153" id="p-153" id="p-153" id="p-153" id="p-153" id="p-153" id="p-153" id="p-153" id="p-153"
[0153] In certain embodiments, the invention contemplates an antibody variant that possesses some but not all effector functions, which make it a desirable candidate for applications in which the half life of the antibody in vivo is important yet certain effector functions (such as ADCC) are unnecessary or deleterious. In vitro and/or in vivo cytotoxicity assays can be conducted to confirm the reduction/depletion of GDC and/or ADCC activities. For example, Fc receptor (FcR) binding assays can be conducted to ensure that the antibody lacks FcyR binding (hence likel lacky ing ADCC activity), but retains FcRn binding ability. The primary cells for mediating ADCC, NK cells, express FcyRIII only, wherea smonocytes express FcyRI, FcyRII and FcyRIII. FcR expression on hematopoietic cells is summarize din Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol., vol. 9, pp. 457-492, 1991. Non- limiting examples of in vitro assays to asses sADCC activity of a molecul ofe interest is described in U.S. Pat. No. 5,500,362 (see also, e.g. Hellstrom et al. Proc. Nat'l Acad. Sci.
USA, vol. 83, pp. 7059-7063, 1986) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA, vol. 82, pp. 1499-1502, 1985; U.S. Pat. No. 5,821,337 (see also Bruggemann et al., J. Exp.
Med., vol. 166, pp. 1351-1361, 1987). Alternatively, non-radioactive assays methods may be employed (see, for example, ACTITM non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, Calif.; and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, Wis.). Useful effector cells for such assays include periphera l blood mononuclea cellsr (PBMC) and Natural Killer (NK) cells. Alternatively or , additionally, ADCC activity of the molecul ofe interest may be assessed in vivo, e.g., in a animal model such as that disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA, vol. 95, pp. 652-656, 1998. Clq binding assays may also be carried out to confirm that the antibody is unable to bind Clq and hence lacks CDC activity. See, e.g., Clq and C3c binding ELISA in WO 2006/029879 and WO 2005/100402. To asses scomplement activation, a CDC assay may be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods, vol. 202, pp.163-171, 1996; Cragg, M. S. et al., Blood, vol. 101, pp. 1045-1052, 2003; and Cragg, M.
S, and M. J. Glennie, Blood, vol. 103, pp. 2738-2743, 2004). FcRn binding and in vivo 38WO 2020/092155 PCT/US2019/058066 clearance/hal liffe determinations can also be performed using methods known in the art (see, e.g., Petkova, S. B. et a\.,Int'l. Immunol., vol. 18, pp. 1759-1769, 2006). 154. 154. id="p-154" id="p-154" id="p-154" id="p-154" id="p-154" id="p-154" id="p-154" id="p-154" id="p-154" id="p-154" id="p-154" id="p-154"
[0154] Antibodies with reduced effector function include those with substitution of one or more of Fc region residue s238, 265, 269, 270, 297, 327 and 329 (U.S. Pat. No. 6,737,056).
Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called "DANA" Fc mutant with substitution of residues 265 and 297 to alanine (U.S. Pat. No. 7,332,581). 155. 155. id="p-155" id="p-155" id="p-155" id="p-155" id="p-155" id="p-155" id="p-155" id="p-155" id="p-155" id="p-155" id="p-155" id="p-155"
[0155] Certain antibody variants with improved or diminished binding to FcRs are described.
(See, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312, and Shields et al., 7. Biol. Chern., vol. 9, pp. 6591-6604, 2001). 156. 156. id="p-156" id="p-156" id="p-156" id="p-156" id="p-156" id="p-156" id="p-156" id="p-156" id="p-156" id="p-156" id="p-156" id="p-156"
[0156] In certain embodiments, an antibody variant comprises an Fc region with one or more amino acid substitutions which improve ADCC, e.g., substitutions at positions 298, 333, and/or 334 of the Fc region (EU numbering of residues). 157. 157. id="p-157" id="p-157" id="p-157" id="p-157" id="p-157" id="p-157" id="p-157" id="p-157" id="p-157" id="p-157" id="p-157" id="p-157"
[0157] In some embodiments alt, erations are made in the Fc region that resul int altered (i.e., either improved or diminished) Clq binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99/51642, and Idusogie et al. 7.
Immunol., vol. 164, pp. 4178-4184, 2000. 158. 158. id="p-158" id="p-158" id="p-158" id="p-158" id="p-158" id="p-158" id="p-158" id="p-158" id="p-158" id="p-158" id="p-158" id="p-158"
[0158] Antibodies with increased half lives and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., 7.
Immunol., vol. 117, pp. 587-593, 1976 and Kim et al., 7. Immunol., vol. 24, p. 249, 1994), are described in US2005/0014934. Those antibodies comprise an Fc region with one or more substitutions therein which improve binding of the Fc region to FcRn. Such Fc variants include/e those with substitutions at one or more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, e.g., substitution of Fc region residue 434 (U.S. Pat. No. 7,371,826). See also Duncan & Winter, Nature, vol. 322, pp. 738-740, 1988; U.S. Pat. No. 5,648,260; U.S. Pat. No. ,624,821; and WO 94/29351 concerning other examples of Fc region variants.
Cysteine Engineered Antibody Variants 159. 159. id="p-159" id="p-159" id="p-159" id="p-159" id="p-159" id="p-159" id="p-159" id="p-159" id="p-159" id="p-159" id="p-159" id="p-159"
[0159] In certain embodiments, it may be desirable to create cysteine engineered antibodies , e.g., "thioMAbs," in which one or more residues of an antibody are substituted with cysteine residues In. particular embodiments, the substituted residues occur at accessibl sitee s of the antibody. By substituting those residues with cysteine, reactive thiol groups are thereby positioned at accessible sites of the antibody and may be used to conjugate the antibody to 39WO 2020/092155 PCT/US2019/058066 other moieties, such as drug moieties or linker-drug moieties, to create an immunoconjugate, as described further herein. In certain embodiments any, one or more of the following residues may be substituted with cysteine: V205 (Kabat numbering) of the light chain; Al 18 (EU numbering) of the heavy chain; and 5400 (EU numbering) of the heavy chain Fc region.
Cysteine engineered antibodies may be generated as described, e.g., in U.S. Pat. No. 7,521,541.
Antibody Derivatives 160. 160. id="p-160" id="p-160" id="p-160" id="p-160" id="p-160" id="p-160" id="p-160" id="p-160" id="p-160" id="p-160" id="p-160" id="p-160"
[0160] In certain embodiments, an antibody or antibody fragment provided herei nmay be further modified to contain additional nonproteinaceous moieties that are known in the art and readily available. The moieties suitable for derivatization of the antibody or antibody fragment include but are not limite dto water soluble polymers. Non-limiting examples of water soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylen glycol,e carboxymethylcellul dextraose, n, polyvinyl alcohol, polyvinyl pyrrolidone, poly-l,3-dioxolane, poly-l,3,6-trioxane, ethylene/mal eic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, propropylene glycol homopolymers , prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyol s(e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer may be of any molecular weight, and may be branched or unbranched. The number of polymers attached to the antibody or antibody fragment may vary, and if more than one polymer are attached, they can be the same or different molecules In. general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limite dto, the particular properties or functions of the antibody or antibody fragment to be improved, whether the derivative wil lbe used in a therapy under defined conditions, etc. 161. 161. id="p-161" id="p-161" id="p-161" id="p-161" id="p-161" id="p-161" id="p-161" id="p-161" id="p-161" id="p-161" id="p-161" id="p-161"
[0161] In another embodiment ,conjugates of an antibody or antibody fragment and nonproteinaceous moiety that may be selectively heate dby exposure to radiation are provided. In one embodiment the, nonproteinaceous moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Set. USA, vol. 102, pp. 11600-11605, 2005). The radiation may be of any wavelength, and includes but, is not limited to, wavelengths that do not harm ordinary cell s, but which heat the nonproteinaceous moiety to a temperatur eat which cell proximals to the antibody-nonproteinaceous moiety are killed. 40WO 2020/092155 PCT/US2019/058066 162. 162. id="p-162" id="p-162" id="p-162" id="p-162" id="p-162" id="p-162" id="p-162" id="p-162" id="p-162" id="p-162" id="p-162" id="p-162"
[0162] In another aspect, the present invention provides an anti-CTLA4 antibody or antibody fragment including the isolated heavy chain variable region polypeptides or isolated light chain variable region polypeptide s.The isolated heavy chain variable region polypeptides comprise the Hl, H2, and H3 regions with SEQ ID NOS: 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38. The isolated light chain variable region polypeptides comprise the LI, L2, andL3 regions with SEQ ID NOS: 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37. 163. 163. id="p-163" id="p-163" id="p-163" id="p-163" id="p-163" id="p-163" id="p-163" id="p-163" id="p-163" id="p-163" id="p-163" id="p-163"
[0163] The anti-CTLA4 antibody or antibody fragment of the invention has a higher binding affinity to CTLA4 under a condition in tumor microenvironment than under a condition in a non-tumor microenvironmen t.In one embodiment ,the condition in tumor microenvironment and the condition in a non-tumor microenvironment are both pH. The anti-CTLA4 antibodies or antibody fragments of the invention thus can selectively bind to CTLA4 at a pH about 5.0- 6.8 but wil lhave a lower binding affinity to CTLA4 at a pH about 7.2-7.8 encountered in a normal physiological environment. As shown Examples 2-3, the anti-CTLA4 antibodies or antibody fragments have higher binding affinity to CTLA4 at pH 6.0 that at pH 7.4. 164. 164. id="p-164" id="p-164" id="p-164" id="p-164" id="p-164" id="p-164" id="p-164" id="p-164" id="p-164" id="p-164" id="p-164" id="p-164"
[0164] In certain embodiments, the anti-CTLA4 antibodies or antibody fragments of the present invention have a dissociation constant (Kd) with CTLA4 under a condition in tumor microenvironment of about ^1 pM, ^100 nM, ^10 nM, ^1 nM, ^0.1 nM, ^0.01 nM, or ^0.001 nM (e.g. 108־M or les s,or from 108־M to 1013־M, or from 109־M to 10-13 M). In one embodiment the, ratio of the Kd of the antibody or antibody fragment with CTLA4 at a value of the condition in tumor microenvironment to the Kd at a different value of the same condition in non-tumor microenvironment is at least about 1.5:1, at least about 2:1, at least about 3:1, at least about 4:1, at least about 5:1, at least about 6:1, at least about 7:1, at least about 8:1, at least about 9:1, at least about 10:1, at leas aboutt 20:1, at least about 30:1, at least about 50:1, at leas aboutt 70:1, or at least about 100:1. 165. 165. id="p-165" id="p-165" id="p-165" id="p-165" id="p-165" id="p-165" id="p-165" id="p-165" id="p-165" id="p-165" id="p-165" id="p-165"
[0165] In one embodiment ,Kd is measured by a radiolabeled antigen binding assay (RIA) performed with the Fab version of an antibody of interest and its antigen using the following assay. Solution binding affinity of Fabs for antigen is measured by equilibrating Fab with a minimal concentration of (125!)-labeled antigen in the presence of a titration series of unlabeled antigen, then capturing bound antigen with an anti-Fab antibody-coated plate (see, e.g., Chen et al., J. Mol. 5zo/.293:865-881 (1999)). To establish conditions for the assay, MICROTITER® multi-well plate s(Therm oScientific) are coated overnight with 5 ug/ml of a capturing anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), and subsequently blocke dwith 2% (w/v) bovine serum albumin in PBS for two to five hours at 41WO 2020/092155 PCT/US2019/058066 room temperature (approximately 23 °C.). In a non-adsorbent plate (Nunc #269620), 100 pM or 26 pM [125!]-antigen are mixed with serial dilutions of a Fab of interest (e.g., consistent with assessment of the anti-VEGF antibody, Fab-12, in Presta et al., Cancer Res. 57:4593- 4599 (1997)). The Fab of interest is then incubated overnight ;however, the incubation may continue for a longer period (e.g., about 65 hours) to ensure that equilibrium is reached .
Thereafte r,the mixtures are transferred to the capture plate for incubation at room temperature (e.g., for one hour). The solution is then removed and the plate washed eight times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. When the plates have dried, 150 ul/wel ofl scintillant (MICROSCINT-20™; Packard) is added, and the plates are counted on a TOPCOUNT™ gamma counter (Packard) for ten minutes. Concentrations of each Fab that give les sthan or equal to 20% of maximal binding are chosen for use in competitive binding assays. 166. 166. id="p-166" id="p-166" id="p-166" id="p-166" id="p-166" id="p-166" id="p-166" id="p-166" id="p-166" id="p-166" id="p-166" id="p-166"
[0166] According to another embodiment, Kd is measured using surface plasmon resonance assays using a BIACORE®-2000 or a BIACORE®-3000 (BIAcore, Inc., Piscataway, N.J.) at ° C. with immobilized antigen CMS chips at about 10 response units (RU). Briefly, carboxymethylated dextran biosensor chips (CMS, BIACORE, Inc.) are activated with N- ethyl-N'-(3-dimethylaminopropyl)-carbodiim hydrochide loride (EDC) and N- hydroxysuccinimide (NHS) according to the supplier's instructions. Antigen is dilute dwith mM sodium acetate, pH 4.8, to 5 ug/ml (0.2־ pM) before injection at a flow rate of 5 ul/minute to achieve approximately 10 response units (RU) of coupled protein. Following the injection of antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetics measurement s,two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with 0.05% polysorbate 20 (TWEEN-20™) surfactant (PBST) at 25° C. at a flow rate of approximatel y25 ul/min. Association rates (kon) and dissociation rates (kOff) are calculate d using a simple one-to-one Langmuir binding model (BIACORE® Evaluation Software version 3.2) by simultaneously fitting the association and dissociation sensorgram s.The equilibrium dissociation constant (Kd) is calculate asd the ratio koff/kon. See, e.g., Chen et al., 7. Mol. Biol. 293:865-881 (1999). If the on-rate exceeds 106M-1 s1־ by the surface plasmon resonance assay above, then the on-rate can be determined by using a fluorescent quenching technique that measures the increase or decrease in fluorescence emission intensity (excitation=295 nm; emission=340 nm, 16 nm band-pass) at 25° C. of a 20 nM anti- antigen antibody (Fab form) in PBS, pH 7.2, in the presence of increasing concentrations of antigen as measured in a spectromete r,such as a stop-flow equipped spectrophometer (Aviv 42WO 2020/092155 PCT/US2019/058066 Instruments) or a 8000-series SLM-AMINCO™ spectrophotometer (ThermoSpectronic wit) h a stirred cuvette. 167. 167. id="p-167" id="p-167" id="p-167" id="p-167" id="p-167" id="p-167" id="p-167" id="p-167" id="p-167" id="p-167" id="p-167" id="p-167"
[0167] The anti-CTLA4 antibodies of the invention may be a chimeric ,humanized or human antibody. In one embodiment ,an anti-CTLA4 antibody fragment is employe d,e.g., a Fv, Fab, Fab', Fab'-SH, scFv, a diabody, a triabody, a tetrabody or an F(ab')2 fragment and multispecifi cantibodies formed from antibody fragments .In another embodiment, the antibody is a full length antibody, e.g., an intact IgG antibody or other antibody class or isotype as defined herein. For a review of certain antibody fragments, see Hudson et al. Nat.
Med., vol. 9, pp. 129-134, 2003. For a review of scFv fragments, see, e.g., Pluckthiin, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer- Verlag, New York), pp. 269-315 (1994); see also WO 93/16185; and U.S. Pat. Nos. ,571,894 and 5,587,458. For discussion of Fab and F(ab')2 fragments comprising salvage receptor binding epitope residues and having increased in vivo half-life, see U.S. Pat. No. ,869,046. 168. 168. id="p-168" id="p-168" id="p-168" id="p-168" id="p-168" id="p-168" id="p-168" id="p-168" id="p-168" id="p-168" id="p-168" id="p-168"
[0168] The diabodies of the invention may be bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Set. USA, vol. 90, pp. 6444-6448, 1993 for examples of diabodies.
Examples of triabodies and tetrabodies are also described in Hudson et al., Nat. Med., vol. 9, pp. 129-134, 2003. 169. 169. id="p-169" id="p-169" id="p-169" id="p-169" id="p-169" id="p-169" id="p-169" id="p-169" id="p-169" id="p-169" id="p-169" id="p-169"
[0169] In some embodiments the, invention comprises single-domai nantibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single-domai nantibody is a human single-domain antibody (Domantis, Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516 Bl). 170. 170. id="p-170" id="p-170" id="p-170" id="p-170" id="p-170" id="p-170" id="p-170" id="p-170" id="p-170" id="p-170" id="p-170" id="p-170"
[0170] Antibody fragments can be made by various techniques incl, uding but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells (e.g. E. coll or phage), as described herein. 171. 171. id="p-171" id="p-171" id="p-171" id="p-171" id="p-171" id="p-171" id="p-171" id="p-171" id="p-171" id="p-171" id="p-171" id="p-171"
[0171] In some embodiments the, anti-CTLA4 antibodies of the invention may be chimeric antibodies. Certain chimeric antibodies are described, e.g., in U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, vol. 81, pp. 6851-6855, 1984). In one example, the chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region. In a further example, the chimeric antibody is a "class switched" antibody in 43WO 2020/092155 PCT/US2019/058066 which the class or subclas sof the antibody has been changed relative to the class or subclas s of the parent antibody. Chimeri cantibodies include antigen-binding fragments thereof. 172. 172. id="p-172" id="p-172" id="p-172" id="p-172" id="p-172" id="p-172" id="p-172" id="p-172" id="p-172" id="p-172" id="p-172" id="p-172"
[0172] In certain embodiments, the chimeric antibody of the invention is a humanized antibody. Typically, such a non-human antibody is humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody.
Generally, a humanized antibody comprises one or more variable domains in which CDRs (or portions thereof )are derived from a non-human antibody, and FRs (or portions thereof) are derived from human antibody sequence s.A humanize dantibody may optionall yalso comprise at least a portion of a human constant region. In some embodiments, some FR residues in a humanize dantibody are substituted with corresponding residues from a non- human antibody (e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve antibody specificity or affinity. 173. 173. id="p-173" id="p-173" id="p-173" id="p-173" id="p-173" id="p-173" id="p-173" id="p-173" id="p-173" id="p-173" id="p-173" id="p-173"
[0173] Humanized antibodies and methods of making them are reviewed, e.g., in Almagro and Fransson, Front. Biosci., vol. 13, pp. 1619-1633, 2008, and are further described, e.g., in Riechmann et al., Nature, vol. 332, pp. 323-329, 1988; Queen et al., Proc. Nat'l Acad. Set.
USA, vol. 86, pp. 10029-10033, 1989; U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods, vol. 36, pp. 25-34, 2005 (describing SDR (a-CDR) grafting); Padlan, Mol. Immunol., vol. 28, pp. 489-498, 1991 (describing "resurfacing"); Dall'Acqua et al., Methods, vol. 36, pp. 43-60, 2005 (describing "FR shuffling"); and Osbourn et al., Methods, vol. 36, pp. 61-68, 2005 and Klimka et al., Br. J. Cancer, vol. 83, pp. 252-260, 2000 (describing the "guided selection" approach to FR shuffling). 174. 174. id="p-174" id="p-174" id="p-174" id="p-174" id="p-174" id="p-174" id="p-174" id="p-174" id="p-174" id="p-174" id="p-174" id="p-174"
[0174] Human framework regions that may be used for humanization include but are not limite dto: framework regions selected using the "best-fit" method (see ,e.g., Sims et al. J.
Immunol., vol. 151, p. 2296, 1993); framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Set. USA, vol. 89, p. 4285, 1992; and Presta et al. J.
Immunol., vol. 151, p. 2623, 1993); human mature (somaticall muty ated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci., vol. 13, pp. 1619-1633, 2008); and framework regions derived from screening FR libraries (see, e.g., Baca et al., J. Biol. Chern., vol. 272, pp. 10678-10684, 1997 and Rosok et al., J. Biol.
Chern., vol. 271, pp. 22611-22618, 1996). 175. 175. id="p-175" id="p-175" id="p-175" id="p-175" id="p-175" id="p-175" id="p-175" id="p-175" id="p-175" id="p-175" id="p-175" id="p-175"
[0175] In some embodiments the, anti-CTLA4 antibodies of the invention are multispecific, e.g. bispecific antibodies .Multispecific antibodies are monoclona lantibodies that have binding specificitie sfor at least two different sites. In certain embodiments, one of the 44WO 2020/092155 PCT/US2019/058066 binding specificitie sis for CTLA4 and the other is for another antigen. In certain embodiments, bispecific antibodies may bind to two different epitopes of CTLA4. Bispecific antibodies may also be used to localize cytotoxic agents to cell whics h expres sCTLA4.
Bispecific antibodies can be prepared as full length antibodies or antibody fragments. 176. 176. id="p-176" id="p-176" id="p-176" id="p-176" id="p-176" id="p-176" id="p-176" id="p-176" id="p-176" id="p-176" id="p-176" id="p-176"
[0176] Techniques for making multispecifi cantibodies include, but are not limite dto, recombinan tco-expression of two immunoglobulin heavy chain-light chain pairs having different specificitie s(see Milstei nand Cuell o,Nature, vol. 305, pp. 537-540, 1983), WO 93/08829, and Traunecker et al., EMBO J. vol. 10, pp. 3655-3659, 1991), and "knob-in-hole" engineering (see ,e.g., U.S. Pat. No. 5,731,168). Multi-specific antibodies may also be made by engineering electrostatic steering effects for making antibody Fc-heterodimeric molecul es (WO 2009/089004A1); cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat.
No. 4,676,980, and Brennan et al., Science, vol. 229, pp. 81-83, 1985); using leucine zippers to produce bi-specific antibodies (see, e.g., Kostelny et al., J. Immunol., vol. 148, pp. 1547- 1553, 1992); using "diabody" technology for making bispecific antibody fragments (see ,e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, vol. 90, pp. 6444-6448, 1993); and using single- chain Fv (scFv) dimers (see, e.g. Gruber et al., J. Immunol., vol. 152, pp. 5368-5374, 1994); and preparing trispecific antibodies as described, e.g., in Tutt et al. J. Immunol., vol. 147, pp. 60-69, 1991. 177. 177. id="p-177" id="p-177" id="p-177" id="p-177" id="p-177" id="p-177" id="p-177" id="p-177" id="p-177" id="p-177" id="p-177" id="p-177"
[0177] Engineered antibodies with three or more functional antigen binding sites, including "Octopus antibodies," are also included herei n(see, e.g. US 2006/0025576A1). 178. 178. id="p-178" id="p-178" id="p-178" id="p-178" id="p-178" id="p-178" id="p-178" id="p-178" id="p-178" id="p-178" id="p-178" id="p-178"
[0178] The anti-CTLA4 antibodies or antibody fragments of the invention may be produced using recombinan tmethods and compositions ,which are described in detail in US 2016/0017040. 179. 179. id="p-179" id="p-179" id="p-179" id="p-179" id="p-179" id="p-179" id="p-179" id="p-179" id="p-179" id="p-179" id="p-179" id="p-179"
[0179] The physical/chemi calproperties and/or biologica lactivities of the anti-CTLA4 antibodies or antibody fragments of the invention may be tested and measured by various assays known in the art. Some of thes eassays are described in U.S. Patent No. 8,853,369.
B. Immunoconjugates 180. 180. id="p-180" id="p-180" id="p-180" id="p-180" id="p-180" id="p-180" id="p-180" id="p-180" id="p-180" id="p-180" id="p-180" id="p-180"
[0180] In another aspect, the invention also provides immunoconjugates comprising an anti- CTLA4 antibody or antibody fragment conjugated to one or more cytotoxic agents ,such as chemotherapeuti agenc ts or drugs, growth inhibitory agents ,toxins (e.g., protein toxins, enzymaticall acty ive toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), or radioactive isotopes. 45WO 2020/092155 PCT/US2019/058066 181. 181. id="p-181" id="p-181" id="p-181" id="p-181" id="p-181" id="p-181" id="p-181" id="p-181" id="p-181" id="p-181" id="p-181" id="p-181"
[0181] In one embodiment the, immunoconjugate is an antibody-drug conjugate (ADC) in which an antibody or antibody fragment is conjugated to one or more drugs, including but not limite dto a maytansinoid (see U.S. Pat. Nos. 5,208,020, 5,416,064 and European Patent EP 0 425 235 Bl); an auristatin such as monomethylauristatin drug moieties DE and DE (MMAE and MMAE) (see U.S. Patent Nos. 5,635,483 and 5,780,588, and 7,498,298); a dolastatin; a calicheamicin or derivative thereof (see U.S. Patent Nos. 5,712,374, 5,714,586, 5,739,116, ,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296; Hinman et al., Cancer Res., vol. 53, pp. 3336-3342, 1993; and Lode et al., Cancer Res., vol. 58, pp. 2925-2928, 1998); an anthracycline such as daunomycin or doxorubicin (see Kratz et al., Current Med. Chem., vol. 13, pp. 477-523, 2006; Jeffrey et al., Bioorganic & Med. Chem. Letters, vol. 16, pp. 358- 362, 2006; Torgov et al., Bioconj. Chem., vol. 16, pp. 717-721, 2005; Nagy et al., Proc. Natl.
Acad. Set. USA, vol. 97, pp. 829-834, 2000; Dubowchik et al., Bioorg. & Med. Chem. Letters, vol. 12, vol. 1529-1532, 2002; King et al., J. Med. Chem., vol. 45, pp. 4336-4343, 2002; and U.S. Pat. No. 6,630,579); methotrexate; vindesine ;a taxane such as docetaxel, paclitaxel, larotaxel, tesetaxel and, ortataxel; a trichothecene and; CC1065. 182. 182. id="p-182" id="p-182" id="p-182" id="p-182" id="p-182" id="p-182" id="p-182" id="p-182" id="p-182" id="p-182" id="p-182" id="p-182"
[0182] In another embodiment ,an immunoconjugate comprises an antibody or antibody fragment as described herein conjugated to an enzymatically active toxin or fragment thereof, including but not limite dto diphtheri aA chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin. Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantiainbibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogelli n,restrictocin, phenomycin, enomycin, and the tricothecenes. 183. 183. id="p-183" id="p-183" id="p-183" id="p-183" id="p-183" id="p-183" id="p-183" id="p-183" id="p-183" id="p-183" id="p-183" id="p-183"
[0183] In another embodiment ,an immunoconjugate comprises an antibody or antibody fragment as described herein conjugated to a radioactive atom to form a radioconjugate. A variety of radioactive isotopes are available for the production of radioconjugates .Examples include At211,1131,1125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212 and radioactive isotopes of Lu. When the radioconjugate is used for detection, it may comprise a radioactive atom for scintigraphic studies, for exampl etc99m or 1123, or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as iodine-123 again, iodine-131, indium-Ill, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron. 184. 184. id="p-184" id="p-184" id="p-184" id="p-184" id="p-184" id="p-184" id="p-184" id="p-184" id="p-184" id="p-184" id="p-184" id="p-184"
[0184] Conjugates of an antibody/antibody fragment and cytotoxic agent may be made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio) 46WO 2020/092155 PCT/US2019/058066 propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane- 1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HC1), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine bis-), diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediam ine),diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro- 2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitettaet al., Science, vol. 238, pp. 1098-, 1987. Carbon-14-labeled 1-isothiocyanatobenzyl - 3-methyldiethyle triamne inepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See WO94/11026. The linke mayr be a "cleavable linke"r facilitating releas ofe a cytotoxic drug in the cell For. example, an acid- labile linker, peptidase-sensitive linker, photolabil line ker, dimethyl linke orr disulfide- containing linke (Charir et al., Cancer Res., vol. 52, pp. 127-131, 1992; U.S. Pat. No. ,208,020) may be used. 185. 185. id="p-185" id="p-185" id="p-185" id="p-185" id="p-185" id="p-185" id="p-185" id="p-185" id="p-185" id="p-185" id="p-185" id="p-185"
[0185] The immunuoconjugate sherein expressly contemplate but, are not limite dto conjugates prepared with cross-linker reagents including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SLAB, SMCC, SMPB, SMPH, sulfo- EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB ,sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoat whice) h are commercially availabl e(e.g., from Pierce Biotechnology, Inc., Rockford, Ilk, U.S.A). 186. 186. id="p-186" id="p-186" id="p-186" id="p-186" id="p-186" id="p-186" id="p-186" id="p-186" id="p-186" id="p-186" id="p-186" id="p-186"
[0186] An exemplary embodiment of an ADC comprises an antibody or antibody fragment (Ab) which targets a tumor cell a, drug moiety (D), and a linke moier ty (L) that attaches Ab to D. In some embodiments the, antibody is attached to the linke moir ety (L) through one or more amino acid residues, such as lysine and/or cysteine. 187. 187. id="p-187" id="p-187" id="p-187" id="p-187" id="p-187" id="p-187" id="p-187" id="p-187" id="p-187" id="p-187" id="p-187" id="p-187"
[0187] An exemplary ADC has Formul aI as Ab-(L-D)p, where p is 1 to about 20. In some embodiments, the number of drug moieties that can be conjugated to an antibody is limited by the number of free cysteine residues. In some embodiments, free cysteine residues are introduced into the antibody amino acid sequence by the methods described herein.
Exemplary ADC of Formul aI include, but are not limite dto, antibodies that have 1, 2, 3, or 4 engineered cysteine amino acids (Lyon et al., Methods in Enzym., vol. 502, pp. 123-138, 2012). In some embodiments, one or more free cysteine residues are already present in an antibody, without the use of engineering, in which case the existing free cysteine residues may be used to conjugate the antibody to a drug. In some embodiments, an antibody is 47WO 2020/092155 PCT/US2019/058066 exposed to reducing conditions prior to conjugation of the antibody in order to generate one or more free cysteine residues. i) Exemplary Linkers 188. 188. id="p-188" id="p-188" id="p-188" id="p-188" id="p-188" id="p-188" id="p-188" id="p-188" id="p-188" id="p-188" id="p-188" id="p-188"
[0188] A "Linker" (L) is a bifunctional or multifunctional moiety that can be used to link one or more moieties such as drug moieties (D) to an antibody or antibody fragment (Ab) to form an immunoconjugate such as an ADC of the Formula I. In some embodiments, ADCs can be prepared using a Linker having reactive functionalities for covalently attaching to the drug and to the antibody. For example, in some embodiments a, cysteine thiol of an antibody or antibody fragment (Ab) can form a bond with a reactive functional group of a linke orr a drug-linker intermediate to make an ADC. 189. 189. id="p-189" id="p-189" id="p-189" id="p-189" id="p-189" id="p-189" id="p-189" id="p-189" id="p-189" id="p-189" id="p-189" id="p-189"
[0189] In one aspect, a linke hasr a functionality that is capable of reacting with a free cysteine present on an antibody to form a covalent bond. Nonlimiting exemplary such reactive functionalitie sinclude maleimide, haloacetamides, a-haloacetyl, activated esters such as succinimide esters 4-nitrophenyl, esters, pentafluorophenyl esters, tetrafluorophenyl esters, anhydrides, acid chlorides sulf, onyl chlorides isocyanates, ,and isothiocyanates See,. e.g., the conjugation method at page 766 of Klussman et, al, Bioconjugate Chemistry, vol. 15, pp. 765-773, 2004. 190. 190. id="p-190" id="p-190" id="p-190" id="p-190" id="p-190" id="p-190" id="p-190" id="p-190" id="p-190" id="p-190" id="p-190" id="p-190"
[0190] In some embodiments a, linke hasr a functionality that is capable of reacting with an electrophili groupc presen ton an antibody. Exemplary such electrophil groupsic include, but are not limite dto, aldehyde and ketone carbonyl groups. In some embodiments, a heteroatom of the reactive functionality of the linke canr react with an electrophili groupc on an antibody and form a covalent bond to an antibody unit. Nonlimiting exemplary such reactive functionalities include, but are not limite dto, hydrazide, oxime, amino, hydrazine , thiosemicarbazone, hydrazine carboxylate, and arylhydrazide. 191. 191. id="p-191" id="p-191" id="p-191" id="p-191" id="p-191" id="p-191" id="p-191" id="p-191" id="p-191" id="p-191" id="p-191" id="p-191"
[0191] A linke mayr comprise one or more linke compor nents. Exemplary linke componer nts include 6-maleimidocaproyl ("MC"), maleimidopropanoyl ("MP"), valine-citrulline ("val- cit" or "vc"), alanine-phenylalanine ("ala-phe"), p-aminobenzyloxycarbonyl (a "PAB"), N- Succinimidyl 4-(2-pyridylthio) pentanoate ("SPP"), and 4-(N-maleimidomethyl)cyclohe xane- 1 carboxylate ("MCC"). Various linke componentr s are known in the art, some of which are described below. 192. 192. id="p-192" id="p-192" id="p-192" id="p-192" id="p-192" id="p-192" id="p-192" id="p-192" id="p-192" id="p-192" id="p-192" id="p-192"
[0192] A linke mayr be a "cleavable linker," facilitating releas ofe a drug. Nonlimiting exemplary cleavable linkers include acid-labile linkers (e.g., comprising hydrazone), protease-sensiti ve(e.g., peptidase-sensitive) linkers photolabil, linkerse or, disulfide­ 48WO 2020/092155 PCT/US2019/058066 containing linkers (Chari et al., Cancer Research, vol. 52, pp. 127-131, 1992; U.S. Pat. No. ,208,020). 193. 193. id="p-193" id="p-193" id="p-193" id="p-193" id="p-193" id="p-193" id="p-193" id="p-193" id="p-193" id="p-193" id="p-193" id="p-193"
[0193] In certain embodiments, a linke hasr the following Formul aII as —Aa—Ww—Yy—, wherein A is a "stretcher unit", and a is an integer from 0 to 1; W is an "amino acid unit", and w is an integer from 0 to 12; Y is a "spacer unit", and y is 0, 1, or 2. An ADC comprising the linke ofr Formul aII has the Formula 1(A): Ab-(Aa—Ww—Yy-D)p, wherei nAb, D, and p are defined as above for Formula I. Exemplary embodiments of such linkers are described in U.S. Pat. No. 7,498,298. 194. 194. id="p-194" id="p-194" id="p-194" id="p-194" id="p-194" id="p-194" id="p-194" id="p-194" id="p-194" id="p-194" id="p-194" id="p-194"
[0194] In some embodiments a, linke componr ent comprises a "stretcher unit" (A) that links an antibody to another linke componr ent or to a drug moiety. Nonlimiting exemplary stretcher units are shown below (wherein the wavy line indicates sites of covalent attachment to an antibody, drug, or additional linke components)r : 195. 195. id="p-195" id="p-195" id="p-195" id="p-195" id="p-195" id="p-195" id="p-195" id="p-195" id="p-195" id="p-195" id="p-195" id="p-195"
[0195] In some embodiments a, linke componr ent comprises an "amino acid unit" (W). In some such embodiments the, amino acid unit allow sfor cleavage of the linker by a protease , thereby facilitating releas ofe the drug from the immunoconjugate upon exposure to intracellula proter ases, such as lysosomal enzymes (Doronina et al., Nat. Biotechnol., vol. 21, pp. 778-784, 2003). Exemplary amino acid units include ,but are not limite dto, dipeptides, tripeptides ,tetrapeptides and, pentapeptides. Exemplary dipeptides include, but are not limite dto, valine-citrulli ne(vc or val-cit), alanine-phenylalanine (af or ala-phe); phenylalanine-lysi (fkne or phe-lys );phenylalanine-homolysi (phe-hone moly s);and N- methyl-valine-citrull (Me-vaine l-cit). Exemplary tripeptides include ,but are not limite dto, 49WO 2020/092155 PCT/US2019/058066 glycine-valine-citrull (gly-val-cine it) and glycine-glycine-glyc ine(gly-gly-gly). An amino acid unit may comprise amino acid residues that occur naturally and/or minor amino acids and/or non-naturally occurring amino acid analogs, such as citrulline Amino acid units can be designed and optimized for enzymatic cleavage by a particular enzyme, for example, a tumor-associated protease, cathepsin B, C and D, or a plasmin protease. 196. 196. id="p-196" id="p-196" id="p-196" id="p-196" id="p-196" id="p-196" id="p-196" id="p-196" id="p-196" id="p-196" id="p-196" id="p-196"
[0196] Typically, peptide-type linker cans be prepared by forming a peptide bond between two or more amino acids and/or peptide fragments. Such peptide bonds can be prepared, for example, according to a liquid phase synthesis method (e.g., E. Schrode rand K. Liibke (1965) "The Peptides", volume 1, pp 76-136, Academic Press). 197. 197. id="p-197" id="p-197" id="p-197" id="p-197" id="p-197" id="p-197" id="p-197" id="p-197" id="p-197" id="p-197" id="p-197" id="p-197"
[0197] In some embodiments a, linke componr ent comprises a "spacer unit" (Y) that links the antibody to a drug moiety, either directly or through a stretcher unit and/or an amino acid unit. A spacer unit may be "self-immolative" or a "non-self-immolative." A "non-self- immolative" space runit is one in which part or all of the spacer unit remains bound to the drug moiety upon cleavage of the ADC. Examples of non-self-immolative spacer units include, but are not limite dto, a glycine spacer unit and a glycine-glycine spacer unit. In some embodiments, enzymatic cleavage of an ADC containing a glycine-glycine spacer unit by a tumor-cell associated protease results in releas ofe a glycine-glycine-drug moiety from the remainder of the ADC. In some such embodiments the, glycine-glycine-drug moiety is subjected to a hydrolysis step in the tumor cell thus, cleaving the glycine-glycine spacer unit from the drug moiety. 198. 198. id="p-198" id="p-198" id="p-198" id="p-198" id="p-198" id="p-198" id="p-198" id="p-198" id="p-198" id="p-198" id="p-198" id="p-198"
[0198] A "self-immolative" spacer unit allows for releas ofe the drug moiety. In certain embodiments, a spacer unit of a linke comprisr es a p-aminobenzyl unit. In some such embodiments, a p-aminobenzyl alcohol is attached to an amino acid unit via an amide bond, and a carbamate, methylcarbamat e,or carbonate is made between the benzyl alcohol and the drug (Hamann et al. Expert Opin. Ther. Patents, vol. 15, pp. 1087-1103, 2005). In some embodiments, the spacer unit comprises p-aminobenzyloxycarbonyl (PAB). In some embodiments, an ADC comprising a self-immolative linke hasr the structure: wherein Q is —C1-C8 alkyl —, O—(C1-C8 alkyl), -halogen, -nitro, or -cyano; m is an integer ranging from 0 to 4; X may be one or more additional spacer units or may be absent ;and p 50WO 2020/092155 PCT/US2019/058066 ranges from 1 to about 20. In some embodiments, p ranges from 1 to 10, 1 to 7, 1 to 5, or 1 to 4. Nonlimiting exemplary X spacer units include: wherein Ri and R2 are independentl sely ected from H and C1-C6 alkyl In. some embodiments, R! and R2 are each —CH3. 199. 199. id="p-199" id="p-199" id="p-199" id="p-199" id="p-199" id="p-199" id="p-199" id="p-199" id="p-199" id="p-199" id="p-199" id="p-199"
[0199] Other examples of self-immolat ivespacers include ,but are not limite dto, aromatic compounds that are electronicall simy ilar to the PAB group, such as 2-aminoimidazol-5- methanol derivatives (U.S. Pat. No. 7,375,078; Hay et al., Bioorg. Med. Chem. Lett., vol. 9, p. 2237-, 1999) and ortho- or para-aminobenzylacetals In. some embodiments spacers, can be used that undergo cyclization upon amide bond hydrolysis, such as substituted and unsubstituted 4-aminobutyric acid amides (Rodrigues et al., Chemistry Biology, vol. 2, pp. 223-, 1995), appropriately substituted bicyclo[2.2.1] and bicyclo[2.2.2] ring systems (Storm et al., J. Amer. Chem. Soc., vol. 94, p. 5815-, 1972) and 2-aminopheny!propioni cacid amides (Amsberry et al, J. Org. Chem., vol. 55, p. 5867, 1990). Linkage of a drug to the a-carbon of a glycine residue is another exampl eof a self-immolat ivespacer that may be useful in ADCs (Kingsbury et al., J. Med. Chem., vol. 27, p.1447, 1984). 200. 200. id="p-200" id="p-200" id="p-200" id="p-200" id="p-200" id="p-200" id="p-200" id="p-200" id="p-200" id="p-200" id="p-200" id="p-200"
[0200] In some embodiments linke, Lr may be a dendritic type linke forr covalent attachment of more than one drug moiety to an antibody through a branching, multifunctional linker moiety (Sun et al. Bioorganic & Medicinal Chemistry Letters, vol. 12, pp. 2213-2215, 2002; Sun et al., Bioorganic & Medicinal Chemistry, vol. 11, pp. 1761-1768, 2003). Dendritic linkers can increase the mola rratio of drug to antibody, i.e. loading, which is related to the potency of the ADC. Thus, where an antibody bears only one reactive cysteine thiol group, a multitude of drug moieties may be attached through a dendritic linker. 201. 201. id="p-201" id="p-201" id="p-201" id="p-201" id="p-201" id="p-201" id="p-201" id="p-201" id="p-201" id="p-201" id="p-201" id="p-201"
[0201] Nonlimiting exemplary linkers are shown below in the context of an ADC of Formula I: 51WO 2020/092155 PCT/US2019/058066 wherein R! and R2 are independentl sely ected from H and C1-C6 alkyl In. some embodiments, R! and R2 are each —CH3.
Phe-Lys-PAB-Ab wherein n is 0 to 12. In some embodiments n, is 2 to 10. In some embodiments n, is 4 to 8. 202. 202. id="p-202" id="p-202" id="p-202" id="p-202" id="p-202" id="p-202" id="p-202" id="p-202" id="p-202" id="p-202" id="p-202" id="p-202"
[0202] Further nonlimiting exemplary ADCs include the structures: 52WO 2020/092155 PCT/US2019/058066 where X is: —(CH),— C (CH,),—, each R is independentl Hy or Ci-Ce alkyl and; n is 1 to 12. 203. 203. id="p-203" id="p-203" id="p-203" id="p-203" id="p-203" id="p-203" id="p-203" id="p-203" id="p-203" id="p-203" id="p-203" id="p-203"
[0203] In some embodiments a, linke isr substituted with groups that modulate solubili ty and/or reactivity. As a nonlimiting example, a charged substituent such as sulfonat e(— SO3 ) or ammonium may increase water solubili tyof the linke reager nt and facilitate the coupling reaction of the linke reager nt with the antibody and/or the drug moiety, or facilitate the coupling reaction of Ab-L (antibody-linke intermedir ate) with D, or D-L (drug-linker intermediate) with Ab, depending on the synthetic route employed to prepare the ADC. In some embodiments, a portion of the linke isr coupled to the antibody and a portion of the linke isr coupled to the drug, and then the Ab-(linker portion)ais coupled to drug-(linke r portion)b to form the ADC of Formul aI. 53WO 2020/092155 PCT/US2019/058066 204. 204. id="p-204" id="p-204" id="p-204" id="p-204" id="p-204" id="p-204" id="p-204" id="p-204" id="p-204" id="p-204" id="p-204" id="p-204"
[0204] The compounds of the invention expressl conty emplate but, are not limite dto, ADCs prepared with the following linke rear gents: bis-maleimido-trioxyethylene glycol (BMPEO), N-(P־maleimidopropyloxy)-N-hydroxy succinimide ester (BMPS), N-(8- maleimidocaproyloxy) succinimide ester (EMCS), N-[y-maleimidobutyryloxy]succinim ide ester (GMBS), 1,6-hexane-bis-vinylsulfone (HBVS), succinimidyl 4-(N- maleimidomethyl)cyclohexane-1 -carboxy-(6-amidocaproate) (LC-SMCC), m- maleimidobenzoyl-N-hydroxysuccinim esteride (MBS), 4-(4-N-Maleimidophenyl)butyric acid hydrazide (MPBH), succinimidyl 3-(bromoacetamido)propionate (SBAP), succinimidyl iodoacetate (SIA), succinimidyl (4-iodoacetyl)aminobenzoa te(SIAB), N-succinimidyl-3-(2- pyridyldithio) propionate (SPDP), N-succinimidyl-4-(2-pyridylthio)pentanoat (SPP),e succinimidyl 4-(N-maleimidomethy!)cyclohexa1 -carboxyne- late (SMCC), succinimidyl 4-(p- maleimidophenyl)butyrate (SMPB), succinimidyl 6-[(beta- maleimidopropionamido)hexanoate] (SMPH), iminothiolane (IT), sulfo-EMCS ,sulfo- GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and succinimidyl-(4-vinylsulfone)benzoate (SVSB), and including bis-maleimide reagents: dithiobismaleimidoethane (DTME), 1,4-Bismaleimidobutan (BMB),e 1,4 Bismaleimidyl-2,3- dihydroxybutane (BMDB), bismaleimidohexane (BMH), bismaleimidoethane (BMOE), BM(PEG)2 (shown below), and BM(PEG)3 (shown below); bifunctional derivatives of imidoesters (such as dimethyl adipimidate HC1), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p- azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p- diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as l,5-difluoro-2,4-dinitrobenzene). In some embodiments, bis-maleimide reagents allow the attachment of the thiol group of a cysteine in the antibody to a thiol-containing drug moiety, linker, or linker-drug intermediate Othe. r functional groups that are reactive with thiol groups include, but are not limited to, iodoacetamide, bromoacetamide, vinyl pyridine, disulfide pyri, dyl disulfide, isocyanate, and isothiocyanate. 205. 205. id="p-205" id="p-205" id="p-205" id="p-205" id="p-205" id="p-205" id="p-205" id="p-205" id="p-205" id="p-205" id="p-205" id="p-205"
[0205] Certain usefu llinke reagentsr can be obtained from various commercial sources ,such as Pierce Biotechnology, Inc. (Rockford, Ilk), Molecular Biosciences Inc. (Boulder, Colo.), or synthesized in accordance with procedures described in the art; for example, in Toki et al., J. Org. Chem., vol. 67, pp. 1866-1872, 2002; Dubowchik, et al., Tetrahedron Letters, vol. 38, pp. 5257-60, 1997; Walker, J. Org. Chem., vol. 60, pp. 5352-5355, 1995; Frisch et al., 54WO 2020/092155 PCT/US2019/058066 Bioconjugate Chem., vol. 7, pp. 180-186, 1995; U.S. Pat. No. 6,214,345; WO 02/088172; US2003130189; US2003096743; WO 03/026577; WO 03/043583; and WO 04/032828. 206. 206. id="p-206" id="p-206" id="p-206" id="p-206" id="p-206" id="p-206" id="p-206" id="p-206" id="p-206" id="p-206" id="p-206" id="p-206"
[0206] Carbon-14-labeled 1 -isothiocyanatobenzyl -3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See, e.g., WO94/11026. ii) Exemplary Drug Moieties 1) Maytansine and Maytansinoids 207. 207. id="p-207" id="p-207" id="p-207" id="p-207" id="p-207" id="p-207" id="p-207" id="p-207" id="p-207" id="p-207" id="p-207" id="p-207"
[0207] In some embodiments an, immunoconjugate comprises an antibody conjugated to one or more maytansinoid molecules Mayt. ansinoids are derivatives of maytansine ,and are mitototic inhibitors which act by inhibiting tubulin polymerization. Maytansine was first isolated from the east African shrub Maytenus serrata (U.S. Patent No. 3,896,111).
Subsequently, it was discovered that certain microbes also produce maytansinoids, such as maytansinol and C-3 maytansinol esters (U.S. Patent No. 4,151,042). Synthetic maytansinoids are disclosed, for example, in U.S. Patent Nos. 4,137,230; 4,248,870; 4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268; 4,308,269; 4,309,428; 4,313,946; 4,315,929; 4,317,821; 4,322,348; 4,331,598; 4,361,650; 4,364,866; 4,424,219; 4,450,254; 4,362,663; and 4,371,533. 208. 208. id="p-208" id="p-208" id="p-208" id="p-208" id="p-208" id="p-208" id="p-208" id="p-208" id="p-208" id="p-208" id="p-208" id="p-208"
[0208] Maytansinoid drug moieties are attractive drug moieties in antibody-drug conjugates because they are: (i) relativel accessy ibl toe prepare by fermentation or chemical modification or derivatization of fermentation products, (ii) amenable to derivatization with functional groups suitable for conjugation through non-disulfide linkers to antibodies ,(iii) stabl ein plasma, and (iv) effective against a variety of tumor cel llines. 209. 209. id="p-209" id="p-209" id="p-209" id="p-209" id="p-209" id="p-209" id="p-209" id="p-209" id="p-209" id="p-209" id="p-209" id="p-209"
[0209] Certain maytansinoids suitable for use as maytansinoid drug moieties are known in the art and can be isolated from natural sources according to known methods or produced using genetic engineering techniques (see ,e.g., Yu et al., PNAS, vol. 99, pp. 7968-7973, 2002). Maytansinoids may also be prepared syntheticall accordingy to known methods. 210. 210. id="p-210" id="p-210" id="p-210" id="p-210" id="p-210" id="p-210" id="p-210" id="p-210" id="p-210" id="p-210" id="p-210" id="p-210"
[0210] Exemplary maytansinoid drug moieties include ,but are not limite dto, those having a modified aromatic ring, such as: C-19-dechloro (U.S. Patent No. 4,256,746) (prepared ,for example, by lithium aluminum hydride reduction of ansamytocin P2); C-20-hydroxy (or C- -demethyl)+/-C-19-dechloro (U.S. Patent Nos. 4,361,650 and 4,307,016) (prepared, for example, by demethylati onusing Streptomyces or Actinomyces or dechlorination using LAH); and C-20-demethoxy, C-20-acyloxy (—OCOR), +/-dechloro (U.S. Patent No. 55WO 2020/092155 PCT/US2019/058066 4,294,757) (prepared, for example, by acylation using acyl chlorides), and those having modifications at othe rpositions of the aromatic ring. 211. 211. id="p-211" id="p-211" id="p-211" id="p-211" id="p-211" id="p-211" id="p-211" id="p-211" id="p-211" id="p-211" id="p-211" id="p-211"
[0211] Exemplary maytansinoid drug moieties also include those having modifications such as: C-9-SH (U.S. Patent No. 4,424,219) (prepared, for example, by the reaction of maytansinol with H2S or P2S5); C-14-alkoxymethyl(demethoxy/CH2OR)(U Pat..S. No. 4,331,598); C-14-hydroxymethyl or acyloxymethyl (CH:OH or CH2OAc) (U.S. Pat. No. 4,450,254) (prepared, for example, from Nocardia); C-15-hydroxy/acyloxy (U.S. Pat. No. 4,364,866) (prepared, for example, by the conversion of maytansinol by Streptomyces)־, C-15- methoxy (U.S. Patent Nos. 4,313,946 and 4,315,929) (for example, isolated from Trewia midlflordy, C-18-N-demethyl (U.S. Patent Nos. 4,362,663 and 4,322,348) (prepared, for example, by the demethylati onof maytansinol by Streptomycesp, and 4,5-deoxy (U.S. Patent No. 4,371,533) (prepared, for example, by the titanium trichloride/LAH reduction of maytansinol). 212. 212. id="p-212" id="p-212" id="p-212" id="p-212" id="p-212" id="p-212" id="p-212" id="p-212" id="p-212" id="p-212" id="p-212" id="p-212"
[0212] Many positions on maytansinoid compounds are useful as the linkage position. For example, an ester linkage may be formed by reaction with a hydroxyl group using conventional coupling techniques In. some embodiments, the reaction may occur at the C-3 position having a hydroxyl group, the C-14 position modified with hydroxymethyl, the C-15 position modified with a hydroxyl group, and the C-20 position having a hydroxyl group. In some embodiments, the linkage is formed at the C-3 position of maytansinol or a maytansinol analogue. 213. 213. id="p-213" id="p-213" id="p-213" id="p-213" id="p-213" id="p-213" id="p-213" id="p-213" id="p-213" id="p-213" id="p-213" id="p-213"
[0213] Maytansinoid drug moieties include those having the structure: | HO 1 ch3o h where the wavy line indicates the covalent attachment of the sulfur atom of the maytansinoid drug moiety to a linke ofr an ADC. Each R may independentl bey H or a C1-C6 alkyl The . alkylene chain attaching the amide group to the sulfur atom may be methanyl, ethanyl, or propyl, i.e., m is 1, 2, or 3 (U.S. Patent No. 633,410; U.S. Patent No. 5,208,020; Chari et 56WO 2020/092155 PCT/US2019/058066 al., Cancer Res., vol. 52, pp. 127-131, 1992; Liu et al., Proc. Nall. Acad. Set. USA, vol. 93, pp. 8618-8623, 1996). 214. 214. id="p-214" id="p-214" id="p-214" id="p-214" id="p-214" id="p-214" id="p-214" id="p-214" id="p-214" id="p-214" id="p-214" id="p-214"
[0214] All stereoisomers of the maytansinoid drug moiety are contemplated for the ADC of the invention, i.e. any combination of R and S configurations at the chiral carbons (U.S.
Patent Nos. 7,276,497; 6,913,748; 6,441,163; 633,410 (RE39151); 5,208,020; Widdison et al (2006) J. Med. Chem. 49:4392-4408. In some embodiments, the maytansinoid drug moiety has the following stereochemistry: 215. 215. id="p-215" id="p-215" id="p-215" id="p-215" id="p-215" id="p-215" id="p-215" id="p-215" id="p-215" id="p-215" id="p-215" id="p-215"
[0215] Exemplary embodiments of maytansinoid drug moieties include ,but are not limited to, DM1; DM3; and DM4, having the structures: 57WO 2020/092155 PCT/US2019/058066 DM3 wherein the wavy line indicates the covalent attachment of the sulfur atom of the drug to a linke (L)r of an antibody-drug conjugate. 216. 216. id="p-216" id="p-216" id="p-216" id="p-216" id="p-216" id="p-216" id="p-216" id="p-216" id="p-216" id="p-216" id="p-216" id="p-216"
[0216] Exemplary antibody-drug conjugates where DM1 is linked through a BMPEO linker to a thiol group of the antibody have the structure and abbreviation: 58WO 2020/092155 PCT/US2019/058066 where Ab is antibody; n is 0, 1, or 2; and p is 1 to about 20. In some embodiments, p is 1 to , p is 1 to 7, p is 1 to 5, or p is 1 to 4. 217. 217. id="p-217" id="p-217" id="p-217" id="p-217" id="p-217" id="p-217" id="p-217" id="p-217" id="p-217" id="p-217" id="p-217" id="p-217"
[0217] Immunoconjugates containing maytansinoids, methods of making the same ,and their therapeuti cuse are disclosed, for example, in U.S. Patent Nos. 5,208,020 and 5,416,064; US 2005/0276812 Al; and European Patent EP 0 425 235 Bl. See also Liu et al., Proc. Natl.
Acad. Set. USA, vol. 93, pp. 8618-8623, 1996; and Chari et al., Cancer Research, vol. 52, pp. 127-131, 1992. 218. 218. id="p-218" id="p-218" id="p-218" id="p-218" id="p-218" id="p-218" id="p-218" id="p-218" id="p-218" id="p-218" id="p-218" id="p-218"
[0218] In some embodiments antibo, dy-maytansinoid conjugates may be prepared by chemicall liynking an antibody to a maytansinoid molecule without significantly diminishing the biologica lactivity of either the antibody or the maytansinoid molecule. See, e.g., U.S.
Patent No. 5,208,020. In some embodiments, ADC with an average of 3-4 maytansinoid molecules conjugated per antibody molecul hase shown efficacy in enhancing cytotoxicity of target cells without negatively affecting the function or solubili tyof the antibody. In some instances, even one molecule of toxin/antibody is expected to enhance cytotoxicity over the use of naked antibody. 219. 219. id="p-219" id="p-219" id="p-219" id="p-219" id="p-219" id="p-219" id="p-219" id="p-219" id="p-219" id="p-219" id="p-219" id="p-219"
[0219] Exemplary linking groups for making antibody-maytansinoid conjugates include, for example, those described herei nand those disclose ind U.S. Patent No. 5,208,020; EP Patent 0 425 235 Bl; Chari et al., Cancer Research, vol. 52, pp. 127-131, 1992; US 2005/0276812 Al ;and US 2005/016993 Al. (2) Auristatins and Dolastatins 220. 220. id="p-220" id="p-220" id="p-220" id="p-220" id="p-220" id="p-220" id="p-220" id="p-220" id="p-220" id="p-220" id="p-220" id="p-220"
[0220] Drug moieties include dolastatins, auristatins, and analogs and derivatives thereof (U.S. Patent Nos. 5,635,483; 5,780,588; 5,767,237; and 6,124,431). Auristatins are derivatives of the marine mollusk compound dolastatin-10. While not intending to be bound by any particular theory, dolastatins and auristatins have been shown to interfere with microtubule dynamics, GTP hydrolysis and, nuclear and cellula divisr ion (Woyke et al., Antimicrob. Agents and Chemother., vol. 45, pp. 3580-3584, 2001) and have anticancer (U.S.
Pat. No. 5,663,149) and antifungal activity (Pettit et al., Antimicrob. Agents Chemother., vol. 42, pp. 2961-2965, 1998). The dolastatin/auristatin drug moiety may be attached to the antibody through the N (amino) terminus or the C (carboxyl) terminus of the peptidic drug moiety (WO 02/088172; Doronina et al., Nature Biotechnology, vol. 21, pp. 778-784, 2003; Francisco et al., Blood, vol. 102, pp. 1458-1465, 2003). 59WO 2020/092155 PCT/US2019/058066 221. 221. id="p-221" id="p-221" id="p-221" id="p-221" id="p-221" id="p-221" id="p-221" id="p-221" id="p-221" id="p-221" id="p-221" id="p-221"
[0221] Exemplary auristatin embodiments include the N-terminus linked monomethylauristati drugn moieties De and De, disclose ind U.S. Patent Nos. 7,498,298 and 7,659,241: wherein the wavy line of De and De indicates the covalent attachment site to an antibody or antibody-linker component, and independentl aty each location: R2is selected from H and C1-C8 alkyl; R3 is selected from H, C1-C8 alkyl C3-C8, carbocycle, aryl, C1-C8 alkyl-aryl. Ci- C8 alkyl-(C3-C8 carbocycle), C3-C8heterocycle and C1-C8 alkyl-(C3-C8 heterocycle); R4is selected from H, C1-C8 alkyl C3-C8, carbocycle, aryl, C1-C8 alkyl-aryl, Ci- C8 alkyl-(C3-C8 carbocycle), C3-C8heterocycle and C1-C8 alkyl-(C3-C8 heterocycle); R5 is selected from H and methyl; or R4 and R5 jointly form a carbocyclic ring and have the formula —(CRaRb)n— wherein Ra and Rb are independentl sely ected from H, C1-C8 alkyl and C3- C8 carbocycle and n is selected from 2, 3, 4, 5 and 6; R6is selected from H and C1-C8 alkyl; R7 is selected from H, C1-C8 alkyl C3-C8, carbocycle, aryl, C1-C8 alkyl-aryl, Ci- C8 alkyl-(C3-C8 carbocycle), C3-C8heterocycle and C1-C8 alkyl-(C3-C8 heterocycle); each R8 is independentl yselected from H, OH, C1-C8 alkyl, C3-Cg carbocycle and O— (C1-C8 alkyl); R9 is selected from H and C1-C8 alkyl; R10 is selected from aryl or C3-C8 heterocycle; Z is O, S, NH, or NR12, wherein R12 is C1-C8 alkyl; R11 is selected from H, C1-C20 alkyl, aryl, C3-Cg heterocycle, —(R13O)m—R14, or — (R13O)m—CH(R15)2; m is an integer ranging from 1-1000; R13 is C2-C8 alkyl; 60WO 2020/092155 PCT/US2019/058066 R14isHorC1-C8alkyl; each occurrence of e is independentl H,y COOH, —(CH2)n—N(R16)2, —(CH2)n— SO3H, or —(CH2)n—SO3—C1-C8 alkyl; each occurrence of e is independentl H,y C1-C8 alkyl or, —(CH2)n—COOH; R18is selected from — C(R8)2—C(R8)2-aryl, —C(R8)2—C(R8)2—(C3־C8 heterocycle ), and —C(R8)2—C(R8)2—(C3-C8 carbocycle); and n is an integer ranging from 0 to 6. 222. 222. id="p-222" id="p-222" id="p-222" id="p-222" id="p-222" id="p-222" id="p-222" id="p-222" id="p-222" id="p-222" id="p-222" id="p-222"
[0222] In one embodiment R, 3, R4 and R7 are independentl isopropyly or sec-butyl and R5 is —H or methyl. In an exemplary embodiment R, 3 and R4 are each isopropyl, R5 is —H, and R7 is sec-butyl. 223. 223. id="p-223" id="p-223" id="p-223" id="p-223" id="p-223" id="p-223" id="p-223" id="p-223" id="p-223" id="p-223" id="p-223" id="p-223"
[0223] In yet another embodiment, R2 and R6 are each methyl, and R9 is —H. 224. 224. id="p-224" id="p-224" id="p-224" id="p-224" id="p-224" id="p-224" id="p-224" id="p-224" id="p-224" id="p-224" id="p-224" id="p-224"
[0224] In still another embodiment, each occurrence of R8 is —OCH3. 225. 225. id="p-225" id="p-225" id="p-225" id="p-225" id="p-225" id="p-225" id="p-225" id="p-225" id="p-225" id="p-225" id="p-225" id="p-225"
[0225] In an exemplary embodiment ,R3 and R4 are each isopropyl, R2 and R6 are each methyl, R5 is —H, R7 is sec-butyl each, occurrence of R8 is —OCH3, and R9 is —H. 226. 226. id="p-226" id="p-226" id="p-226" id="p-226" id="p-226" id="p-226" id="p-226" id="p-226" id="p-226" id="p-226" id="p-226" id="p-226"
[0226] In one embodiment Z, is —O— or —NH—. 227. 227. id="p-227" id="p-227" id="p-227" id="p-227" id="p-227" id="p-227" id="p-227" id="p-227" id="p-227" id="p-227" id="p-227" id="p-227"
[0227] In one embodiment R, 10 is aryl. 228. 228. id="p-228" id="p-228" id="p-228" id="p-228" id="p-228" id="p-228" id="p-228" id="p-228" id="p-228" id="p-228" id="p-228" id="p-228"
[0228] In an exemplary embodiment ,R10 is -phenyl. 229. 229. id="p-229" id="p-229" id="p-229" id="p-229" id="p-229" id="p-229" id="p-229" id="p-229" id="p-229" id="p-229" id="p-229" id="p-229"
[0229] In an exemplary embodiment ,when Z is —O—, R11 is —H, methyl or t-butyl. 230. 230. id="p-230" id="p-230" id="p-230" id="p-230" id="p-230" id="p-230" id="p-230" id="p-230" id="p-230" id="p-230" id="p-230" id="p-230"
[0230] In one embodiment when, Z is —NH, R11 is —CH(R15)2, wherein R15 is —(CH2)n— N(R16)2, and R16 is —C1-C8 alkyl or — (CH2)n—COOH. 231. 231. id="p-231" id="p-231" id="p-231" id="p-231" id="p-231" id="p-231" id="p-231" id="p-231" id="p-231" id="p-231" id="p-231" id="p-231"
[0231] In another embodiment ,when Z is —NH, R11 is —CH(R15)2, wherein R15 is — (CH2)n—SO3H. 232. 232. id="p-232" id="p-232" id="p-232" id="p-232" id="p-232" id="p-232" id="p-232" id="p-232" id="p-232" id="p-232" id="p-232" id="p-232"
[0232] An exemplary auristatin embodiment of formula De is MMAE, wherein the wavy line indicates the covalent attachment to a linke (L)r of an antibody-drug conjugate: XIMAE 233. 233. id="p-233" id="p-233" id="p-233" id="p-233" id="p-233" id="p-233" id="p-233" id="p-233" id="p-233" id="p-233" id="p-233" id="p-233"
[0233] An exemplary auristatin embodiment of formula De is MMAF, wherein the wavy line indicates the covalent attachment to a linke (L)r of an antibody-drug conjugate: MMAF 61WO 2020/092155 PCT/US2019/058066 234. 234. id="p-234" id="p-234" id="p-234" id="p-234" id="p-234" id="p-234" id="p-234" id="p-234" id="p-234" id="p-234" id="p-234" id="p-234"
[0234] Other exemplary embodiments include monomethylvaline compounds having phenylalani necarboxy modifications at the C-terminus of the pentapeptide auristatin drug moiety (WO 2007/008848) and monomethylvalin compoundse having phenylalani ne sidechai nmodifications at the C-terminus of the pentapeptide auristatin drug moiety (WO 2007/008603). 235. 235. id="p-235" id="p-235" id="p-235" id="p-235" id="p-235" id="p-235" id="p-235" id="p-235" id="p-235" id="p-235" id="p-235" id="p-235"
[0235] Nonlimiting exemplary embodiments of ADCs of Formul aI comprising MMAF and various linke compor nents further include Ab-MC-PAB-MMAF and Ab-PAB-MMAF.
Immunoconjugates comprising MMAF attached to an antibody by a linke thatr is not proteolytical lycleavable have been shown to possess activity comparable to immunoconjugates comprising MMAF attached to an antibody by a proteolytically cleavabl e linke (Doroninar et al., Bioconjugate Chem., vol. 17, pp. 114-124, 2006). In some such embodiments, drug releas ise believed to be affected by antibody degradation in the cell. 236. 236. id="p-236" id="p-236" id="p-236" id="p-236" id="p-236" id="p-236" id="p-236" id="p-236" id="p-236" id="p-236" id="p-236" id="p-236"
[0236] Typically, peptide-base ddrug moieties can be prepared by forming a peptide bond between two or more amino acids and/or peptide fragments. Such peptide bonds can be prepared, for example, according to a liquid phase synthesis method (see, e.g., E. Schrode r and K. Liibke ,"The Peptides", volume 1, pp 76-136, 1965, Academic Press).
Auristatin/dolastatin drug moieties may, in some embodiments, be prepared according to the methods of: U.S. Patent Nos. 7,498,298; 5,635,483; 5,780,588; Pettit et al., 7. Am. Chem.
Soc.,no\. lll,pp. 5463-5465, 1998; Pettit et al., Anti-Cancer Drug Design, vol. 13, pp. 243- 277, 1998; Pettit et al., Synthesis,voL 6, pp. 719-725, 1996; Pettit et al., 7. Chem. Soc. Perkin Trans, vol. 15, pp. 859-863, 1996; and Doronina , Nat. Biotechnol., vol. 21, pp. 778-784, 2003. 237. 237. id="p-237" id="p-237" id="p-237" id="p-237" id="p-237" id="p-237" id="p-237" id="p-237" id="p-237" id="p-237" id="p-237" id="p-237"
[0237] In some embodiments aurist, atin/dolastatin drug moieties of formulas De such as MMAE, and De, such as MMAF, and drug-linker intermediates and derivatives thereof, such as MC-MMAF, MC-MMAE, MC-vc-PAB-MMAF, and MC-vc-PAB-MMAE, may be prepared using methods described in U.S. Patent No. 7,498,298; Doronina et al., Bioconjugate Chem., vol. 17, pp. 114-124, 2006; and Doronina et al., Nat. Biotech., vol. 21, pp. 778-784, 2003 and then conjugated to an antibody of interest. (3) Calicheamicin 238. 238. id="p-238" id="p-238" id="p-238" id="p-238" id="p-238" id="p-238" id="p-238" id="p-238" id="p-238" id="p-238" id="p-238" id="p-238"
[0238] In some embodiments the, immunoconjugate comprise san antibody or antibody fargment conjugated to one or more calicheamici molecn ules The. calicheamic infamily of antibiotics, and analogues thereof, are capable of producing double-stranded DNA breaks at sub-picomolar concentrations (Hinman et al., Cancer Research, vol. 53, pp. 3336-3342, 62WO 2020/092155 PCT/US2019/058066 1993; Lode et al., Cancer Research, vol. 58, pp. 2925-2928, 1998). Calicheamici hasn intracellula siter s of action but, in certain instances, does not readily cross the plasm a membrane Theref. ore, cellul uptakear of these agents through antibody-mediated internalizatio nmay, in some embodiments, greatly enhance their cytotoxic effects.
Nonlimiting exemplary methods of preparing antibody-drug conjugates with a calicheamic in drug moiety are described, for example, in U.S. Patent Nos. 5,712,374; 5,714,586; 5,739,116; and 5,767,285. (4) Pyrrolobenzodiazepines 239. 239. id="p-239" id="p-239" id="p-239" id="p-239" id="p-239" id="p-239" id="p-239" id="p-239" id="p-239" id="p-239" id="p-239" id="p-239"
[0239] In some embodiments an, ADC comprises a pyrrolobenzodiazepine (PBD). In some embodiments, PDB dimers recognize and bind to specific DNA sequences. The natural product anthramycin, a PBD, was first reported in 1965 (Leimgruber et al., J. Am. Chem.
Soc., vol. 87, pp. 5793-5795, 1965; Leimgruber et al., J. Am. Chem. Soc., vol. 87, pp. 5791- 5793, 1965). Since then, a number of PBDs, both naturally-occurring and analogues, have been reported (Thurston et al., Chem. Rev. vol. 1994, pp. 433-465 1994, including dimers of the tricyclic PBD scaffold (U.S. Patent Nos. 6,884,799; 7,049,311; 7,067,511; 7,265,105; 7,511,032; 7,528,126; and 7,557,099). Without intending to be bound by any particular theory, it is believed that the dimer structure imparts the appropriate three-dimension shape al for isohelicit withy the minor groove of B-form DNA, leading to a snug fit at the binding site (Kohn, In Antibiotics III. Springer-Verlag, New York, pp. 3-11 (1975); Hurley and Needham-VanDevanter, Acc. Chem. Res., vol. 19, pp. 230-237, 1986). Dimeric PBD compounds bearing C2 aryl substituent shave been shown to be usefu las cytotoxic agents (Hartley et al Cancer Res., vol. 70, pp. 6849-6858, 2010; Antonow, J. Med. Chem.vol. 53, pp. 2927-2941, 2010; Howard et al., Bioorganic and Med. Chem. Letters, vol. 19, pp. 6463-6466, 2009). 240. 240. id="p-240" id="p-240" id="p-240" id="p-240" id="p-240" id="p-240" id="p-240" id="p-240" id="p-240" id="p-240" id="p-240" id="p-240"
[0240] PBD dimers have been conjugated to antibodies and the resulting ADC shown to have anti-cancer properties. Nonlimiting exemplary linkage sites on the PBD dimer include the five-membered pyrrolo ring, the tether between the PBD units, and the N10-C11 imine group (WO 2009/016516; US 2009/304710; US 2010/047257; US 2009/036431; US 2011/0256157; WO 2011/130598). 241. 241. id="p-241" id="p-241" id="p-241" id="p-241" id="p-241" id="p-241" id="p-241" id="p-241" id="p-241" id="p-241" id="p-241" id="p-241"
[0241] Nonlimiting exemplary PBD dimer components of ADCs are of: 63WO 2020/092155 PCT/US2019/058066 and salts and solvates thereof, wherein: the wavy line indicates the covalent attachment site to the linker; the dotted lines indicate the optional presence of a double bond between Cl and C2 or C2 and C3; R2is independentl sely ected from H, OH, =0, =CH2, CN, R, OR, =CH—RD, =C(Rd )2, O—SO2—R, CO2R and COR, and optionally further selected from halo or dihalo, wherein RD is independently selected from R, CO2R, COR, CHO, CO2H, and halo; R6 and R9 are independentl yselected from H, R, OH, OR, SH, SR, NH2, NHR, NRR', NO2, Me3Sn and halo; R7is independentl sely ected from H, R, OH, OR, SH, SR, NH2, NHR, NRR׳, NO2, Me3Sn and halo; Q is independently selected from O, S and NH; R11 is either H, or R or, where Q is O, SO3M, where M is a metal cation; R and R' are each independently selected from optionall ysubstituted C1-8 alkyl Ci-, 12 alkyl C3-8, heterocyclyl C3-20, heterocycle, and C5-20 aryl groups, and optionally in relation to the group NRR', R and R' together with the nitrogen atom to which they are attached form an optionally substituted 4-, 5-, 6- or 7-membered heterocycli ring; c R12, R16, R19 and R17 are as defined for R2, R6, R9 and R7 respectively; R" is a C3-12 alkyle negroup, which chain may be interrupted by one or more heteroatoms, e.g. O, S, N(H), NMe and/or aromatic rings, e.g. benzene or pyridine, which rings are optionally substituted; and X and X' are independentl yselected from O, S and N(H). 242. 242. id="p-242" id="p-242" id="p-242" id="p-242" id="p-242" id="p-242" id="p-242" id="p-242" id="p-242" id="p-242" id="p-242" id="p-242"
[0242] In some embodiments R, and R' are each independentl yselected from optionally substituted Cm2 alkyl C3-20, heterocycl e,and C5-20 aryl groups, and optionall yin relation to the group NRR', R and R' together with the nitrogen atom to which they are attached form an optionall ysubstituted 4-, 5-, 6- or 7-membered heterocycli ring.c In some embodiments, R9 and R19 are H. In some embodiments, R6 and R16 are H. 64WO 2020/092155 PCT/US2019/058066 243. 243. id="p-243" id="p-243" id="p-243" id="p-243" id="p-243" id="p-243" id="p-243" id="p-243" id="p-243" id="p-243" id="p-243" id="p-243"
[0243] In some embodiments R, 7 are R17 are both OR7A, where R7A is optionally substitute d C1-4 alkyl In. some embodiments, R7Ais Me. In some embodiments, R7Ais Ch2Ph, where Ph is a phenyl group. In some embodiments, X is O. In some embodiments, R11 is H. In some embodiments, there is a double bond between C2 and C3 in each monomer unit. 244. 244. id="p-244" id="p-244" id="p-244" id="p-244" id="p-244" id="p-244" id="p-244" id="p-244" id="p-244" id="p-244" id="p-244" id="p-244"
[0244] In some embodiments R, 2 and R12 are independentl yselected from H and R. In some embodiments, R2 and R12are independentl R.y In some embodiments, R2 and R12 are independently optionall ysubstituted C5-20 aryl or C5-?aryl or C8-10 aryl .In some embodiments, R2 and R12 are independentl optionaly ly substituted phenyl, thienyl, napthyl pyridyl, , quinolinyl, or isoquinolinyl. In some embodiments, R2 and R12 are independentl selecy ted from =0, =CH2, =CH—RD, and =C(RD)2. In some embodiments, R2 and R12 each =CH2. In some embodiments, R2 and R12 are each H. In some embodiments, R2 and R12 are each =0. In some embodiments, R2 and R12 are each =CF2. In some embodiments, R2 and/or R12 are independently =C(RD)2. In some embodiments, R2 and/or R12are independentl =CHy —RD. 245. 245. id="p-245" id="p-245" id="p-245" id="p-245" id="p-245" id="p-245" id="p-245" id="p-245" id="p-245" id="p-245" id="p-245" id="p-245"
[0245] In some embodiments when, R2 and/or R12 is =CH—RD, each group may independently have eithe rconfiguration shown below: In some embodiments, a =CH—RDis in configuration (I). In some embodiments R", is a C3 alkyle negroup or a C5 alkylene group. 246. 246. id="p-246" id="p-246" id="p-246" id="p-246" id="p-246" id="p-246" id="p-246" id="p-246" id="p-246" id="p-246" id="p-246" id="p-246"
[0246] The linkers of PBD dimer-val-cit-PAB-Ab and the PBD dimer-Phe-Lys-PAB-A areb protease cleavable whil, ethe linke ofr PBD dimer-maleimide-acetal is acid-labile. 247. 247. id="p-247" id="p-247" id="p-247" id="p-247" id="p-247" id="p-247" id="p-247" id="p-247" id="p-247" id="p-247" id="p-247" id="p-247"
[0247] PBD dimers and ADCs comprising PBD dimers may be prepared according to methods known in the art. See, e.g., WO 2009/016516; US 2009/304710; US 2010/047257; US 2009/036431; US 2011/0256157; WO 2011/130598. 65WO 2020/092155 PCT/US2019/058066 (5) Anthracyclines 248. 248. id="p-248" id="p-248" id="p-248" id="p-248" id="p-248" id="p-248" id="p-248" id="p-248" id="p-248" id="p-248" id="p-248" id="p-248"
[0248] In some embodiments an, ADC may comprise anthracycline Ant. hracyclines are antibiotic compounds that exhibit cytotoxic activity. While not intending to be bound by any particular theory, studies have indicated that anthracyclines may operate to kill cells by a number of different mechanisms includi, ng: 1) intercalation of the drug molecules into the DNA of the cel lthereby inhibiting DNA-dependent nucleic acid synthesis 2); production by the drug of free radicals which then react with cellula macr romolecules to cause damage to the cells, and/or 3) interactions of the drug molecule wits h the cel lmembrane (see, e.g., C.
Peterson et al., "Transport And Storage Of Anthracycline In Experimental System sAnd Human Leukemia" in Anthracycline Antibiotics In Cancer Therapy; N. R. Bachur, "Free Radical Damage" id. at pp. 97-102). Because of their cytotoxic potential anthracyclines have been used in the treatment of numerous cancers such as leukem ia,breast carcinoma, lung carcinoma, ovarian adenocarcinoma and sarcomas (see e.g., P. H-Wiernik, in Anthracycline: Current Status And New Developments, p. 11). 249. 249. id="p-249" id="p-249" id="p-249" id="p-249" id="p-249" id="p-249" id="p-249" id="p-249" id="p-249" id="p-249" id="p-249" id="p-249"
[0249] Nonlimiting exemplary anthracyclines include doxorubicin, epirubicin, idarubicin, daunomycin, nemorubicin, and derivatives thereof. Immunoconjugates and prodrugs of daunorubicin and doxorubicin have been prepared and studied (Kratz et al., Current Med.
Chem., vol. 13, pp. 477-523, 2006; Jeffrey et al., Bioorganic & Med. Chem. Letters, vol. 16, pp. 358-362. 1996; Torgov et al., Bioconj. Chem., vol. 16, pp. 717-721, 2005; Nagy et al., Proc. Natl. Acad. Sci. USA, vol. 97, pp. 829-834, 2000; Dubowchik et al., Bioorg. & Med.
Chem. Letters, vol. 12, pp. 1529-1532, 2002; King et al., J. Med. Chem., vol. 45, pp. 4336- 4343, 2002; EP 0328147; U.S. Pat. No. 6,630,579). The antibody-drug conjugate BR96- doxorubicin reacts specifically with the tumor-associated antigen Lewis-Y and has been evaluated in phase I and II studies (Sale het al., J. Clin. Oncology, vol. 18, pp. 2282-2292, 2000; Ajani et al., Cancer Jour., vol. 6, pp. 78-81, 2000; Tolcher et al., J. Clin. Oncology, vol. 17, pp. 478-484, 1999). 250. 250. id="p-250" id="p-250" id="p-250" id="p-250" id="p-250" id="p-250" id="p-250" id="p-250" id="p-250" id="p-250" id="p-250" id="p-250"
[0250] PNU-159682 is a potent metabolite (or derivative) of nemorubicin (Quintieri et al., Clinical Cancer Research, vol. 11, pp. 1608-1617, 2005). Nemorubicin is a semisynthet ic analog of doxorubicin with a 2-methoxymorpholino group on the glycoside amino of doxorubicin and has been under clinical evaluation (Grandi et al. Cancer Treat. Rev. vol. 17, pp. 133-138, 1990; Ripamonti et al. Brit. J. Cancer, vol. 65, pp. 703-707, 1992), including phase II/III trials for hepatocellula carcr inoma (Sun et al., Proceedings of the American Society for Clinical Oncology, vol. 22, Absl448, 2003; Quintieri, Proceedings of the 66WO 2020/092155 PCT/US2019/058066 American Association of Cancer Research, vol. 44:1st Ed, Abs 4649, 2003; Pacciarini et al., Jour. Clin. Oncology, vol. 24, p. 14116, 2006). 251. 251. id="p-251" id="p-251" id="p-251" id="p-251" id="p-251" id="p-251" id="p-251" id="p-251" id="p-251" id="p-251" id="p-251" id="p-251"
[0251] Anthracyclines, including PNU-159682, may be conjugated to antibodies through several linkage sites and a variety of linkers (US 2011/0076287; WO2009/099741; US 2010/0034837; WO 2010/009124), including the linkers described herein. 252. 252. id="p-252" id="p-252" id="p-252" id="p-252" id="p-252" id="p-252" id="p-252" id="p-252" id="p-252" id="p-252" id="p-252" id="p-252"
[0252] The linke ofr PNU-159682 maleimide acetal-Ab is acid-labile while, the linkers of PNU-159682-val-cit-PAB-Ab, PNU-159682-val-cit-PAB-spacer-Ab, and PNU-159682-val - cit-PAB-spacer(RR2)-Ab are protease cleavable. (6) Other Drug Moieties 253. 253. id="p-253" id="p-253" id="p-253" id="p-253" id="p-253" id="p-253" id="p-253" id="p-253" id="p-253" id="p-253" id="p-253" id="p-253"
[0253] Drug moieties also include geldanamycin (Mandle ret al., J. Nat. Cancer Inst., vol. 92, pp. 1573-1581, 2000; Mandle ret al., Bioorganic & Med. Chem. Letters, vol. 10, pp. 1025-1028, 2000; Mandler et al., Bioconjugate Chem., vol. 13, pp. 786-791, 2002); and enzymaticall acty ive toxins and fragments thereof, including, but not limite dto, diphtheri aA chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP- S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin , mitogelli n,restrictocin, phenomycin, enomycin and the tricothecene s.See, e.g., WO 93/21232. 254. 254. id="p-254" id="p-254" id="p-254" id="p-254" id="p-254" id="p-254" id="p-254" id="p-254" id="p-254" id="p-254" id="p-254" id="p-254"
[0254] Drug moieties also include compounds with nucleolyti actc ivity (e.g., a ribonuclease or a DNA endonuclease). 255. 255. id="p-255" id="p-255" id="p-255" id="p-255" id="p-255" id="p-255" id="p-255" id="p-255" id="p-255" id="p-255" id="p-255" id="p-255"
[0255] In certain embodiments, an immunoconjugate may comprise a highly radioactive atom. A variety of radioactive isotopes are availabl efor the production of radioconjugated antibodies. Examples include At211,1131,1125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212 and radioactive isotopes of Lu. In some embodiments, when an immunoconjugate is used for detection, it may comprise a radioactive atom for scintigraphic studies, for exampl eTc" or I123, or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as zirconium-89, iodine-123, iodine-131, indium-Ill, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron. Zirconium- 89 may be complexed to various metal chelating agents and conjugated to antibodies ,e.g., for PET imaging (WO 2011/056983). 256. 256. id="p-256" id="p-256" id="p-256" id="p-256" id="p-256" id="p-256" id="p-256" id="p-256" id="p-256" id="p-256" id="p-256" id="p-256"
[0256] The radio- or other label mays be incorporated in the immunoconjugate in known ways. For example, a peptide may be biosynthesized or chemicall synthesy ize usingd suitable 67WO 2020/092155 PCT/US2019/058066 amino acid precursors comprising, for example, one or more fluorine-19 atoms in place of one or more hydrogens .In some embodiments, labels such as Tc", I123, Re186, Re188 and In111 can be attached via a cysteine residue in the antibody. In some embodiments yttri, um-90 can be attached via a lysine residue of the antibody. In some embodiments, the IODOGEN method (Fraker et al., Biochem. Biophys. Res. Commun., vol. 80, pp. 49-57, 1978) can be used to incorporate iodine-123. "Monoclonal Antibodies in Immunoscintigraphy" (Chatal, CRC Press 1989) describes certain other methods. 257. 257. id="p-257" id="p-257" id="p-257" id="p-257" id="p-257" id="p-257" id="p-257" id="p-257" id="p-257" id="p-257" id="p-257" id="p-257"
[0257] In certain embodiments, an immunoconjugate may comprise an antibody conjugated to a prodrug-activating enzyme. In some such embodiments, a prodrug-activating enzyme converts a prodrug (e.g., a peptidyl chemotherapeuti agent,c see WO 81/01145) to an active drug, such as an anti-cancer drug. Such immunoconjugates are useful, in some embodiments, in antibody-dependent enzyme-mediated prodrug therapy ("ADEPT"). Enzymes that may be conjugated to an antibody include, but are not limite dto, alkaline phosphatase s,which are usefu lfor converting phosphate-containing prodrugs into free drugs; arylsulfatase whics, h are usefu lfor converting sulfate-containing prodrugs into free drugs; cytosine deaminase, which is useful for converting non-toxic 5-fluorocytosine into the anti-cancer drug, 5-fluorouracil ; proteases such, as serratia protease, thermolysi subtils, isin, carboxypeptidases and cathepsins (such as cathepsins B and L), which are useful for converting peptide-containing prodrugs into free drugs; D-alanylcarboxypeptidases, which are useful for converting prodrugs that contain D-amino acid substituents; carbohydrate-cleaving enzymes such as P־galactosidase and neuraminidase, which are useful for converting glycosylated prodrugs into free drugs; P־ lactamase, which is useful for converting drugs derivatized with P־lactam sinto free drugs; and penicill inamidases, such as penicill inV amidase and penicillin G amidase, which are usefu lfor converting drugs derivatized at their amine nitrogens with phenoxyacetyl or phenylacetyl groups, respectively, into free drugs. In some embodiments enzym, es may be covalently bound to antibodies by recombinant DNA technique swel lknown in the art. See, e.g., Neuberger et al., Nature, vol. 312, pp. 604-608, 1984. iii) Drug Loading 258. 258. id="p-258" id="p-258" id="p-258" id="p-258" id="p-258" id="p-258" id="p-258" id="p-258" id="p-258" id="p-258" id="p-258" id="p-258"
[0258] Drug loading is represented by p, the average number of drug moieties per antibody in a molecul ofe Formul aI. Drug loading may range from 1 to 20 drug moieties (D) per antibody. ADCs of Formul aI include collections of antibodies conjugated with a range of drug moieties, from 1 to 20. The average number of drug moieties per antibody use in the preparation of ADCs from conjugation reactions may be characterized by conventional 68WO 2020/092155 PCT/US2019/058066 means such as mass spectroscopy, ELISA assay, and HPLC. The quantitative distribution of ADCs in terms of p may also be determined. In some instances, separation, purification, and characterization of homogeneous ADCs where p is a certain value from ADCs with other drug loadings may be achieved by means such as reverse phase HPLC or electrophoresis . 259. 259. id="p-259" id="p-259" id="p-259" id="p-259" id="p-259" id="p-259" id="p-259" id="p-259" id="p-259" id="p-259" id="p-259" id="p-259"
[0259] For some antibody-drug conjugates ,p may be limite dby the number of attachment sites on the antibody. For example, where the attachment is a cysteine thiol, as in certain exemplary embodiments above, an antibody may have only one or severa lcysteine thiol groups, or may have only one or several sufficientl yreactive thiol groups through which a linke mayr be attached. In certain embodiments, higher drug loading, e.g. p>5, may cause aggregation, insolubility toxic, ity, or loss of cellula permr eability of certain antibody-drug conjugates. In certain embodiments, the average drug loading for an ADC ranges from 1 to about 8; from about 2 to about 6; or from about 3 to about 5. Indeed, it has been shown that for certain ADCs, the optimal ratio of drug moieties per antibody may be less than 8, and may be about 2 to about 5 (U.S. Pat. No. 7,498,298). 260. 260. id="p-260" id="p-260" id="p-260" id="p-260" id="p-260" id="p-260" id="p-260" id="p-260" id="p-260" id="p-260" id="p-260" id="p-260"
[0260] In certain embodiments, fewe rthan the theoretical maximum of drug moieties are conjugated to an antibody during a conjugation reaction. An antibody may contain, for example, lysin eresidues that do not react with the drug-linker intermediate or linke reagent,r as discussed below. Generall y,antibodies do not contain many free and reactive cysteine thiol groups which may be linked to a drug moiety; indeed most cysteine thiol residues in antibodies exis tas disulfide bridges. In certain embodiments, an antibody may be reduced with a reducing agent such as dithiothreitol (DTT) or tricarbonylethylphosphine (TCEP), under partial or total reducing conditions, to generate reactive cysteine thiol groups. In certain embodiments, an antibody is subjected to denaturing conditions to reveal reactive nucleophili groupsc such as lysin eor cysteine. 261. 261. id="p-261" id="p-261" id="p-261" id="p-261" id="p-261" id="p-261" id="p-261" id="p-261" id="p-261" id="p-261" id="p-261" id="p-261"
[0261] The loading (drug/antibody ratio) of an ADC may be controlled in different ways, and for example, by: (i) limiting the mola rexcess of drug-linker intermediate or linke reagentr relative to antibody, (ii) limiting the conjugation reaction time or temperature, and (iii) partial or limiting reductive conditions for cysteine thiol modification. 262. 262. id="p-262" id="p-262" id="p-262" id="p-262" id="p-262" id="p-262" id="p-262" id="p-262" id="p-262" id="p-262" id="p-262" id="p-262"
[0262] It is to be understood that where more than one nucleophilic group reacts with a drug- linke interr mediate or linker reagent, then the resulting product is a mixture of ADCs with a distribution of one or more drug moieties attached to an antibody. The average number of drugs per antibody may be calculate fromd the mixture by a dual ELISA antibody assay, which is specific for antibody and specific for the drug. Individual ADCs may be identified in the mixture by mass spectroscopy and separated by HPLC, e.g. hydrophobic interaction 69WO 2020/092155 PCT/US2019/058066 chromatography (see ,e.g., McDonagh et al., Prot. Engr. Design & Selection, vol. 19, pp. 299-307, 2006; Hamblett et al., Clin. Cancer Res., vol. 10, pp. 7063-7070, 2004). In certain embodiments, a homogeneous ADC with a singl eloading value may be isolated from the conjugation mixture by electrophoresi ors chromatography. iv) Certain Methods of Preparing Immunoconjugates 263. 263. id="p-263" id="p-263" id="p-263" id="p-263" id="p-263" id="p-263" id="p-263" id="p-263" id="p-263" id="p-263" id="p-263" id="p-263"
[0263] An immunoconjugate that is an ADC of Formul aI may be prepared by severa lroutes employing organic chemistry reactions, conditions, and reagents known to those skill edin the art, including: (1) reaction of a nucleophilic group of an antibody with a bivalent linker reagent to form Ab-L via a covalent bond, followed by reaction with a drug moiety D; and (2) reaction of a nucleophili groupc of a drug moiety with a bivalent linke reagent,r to form D-L, via a covalent bond, followed by reaction with a nucleophilic group of an antibody.
Exemplary methods for preparing an ADC of Formula I via the latter route are described in U.S. Patent No. 7,498,298. 264. 264. id="p-264" id="p-264" id="p-264" id="p-264" id="p-264" id="p-264" id="p-264" id="p-264" id="p-264" id="p-264" id="p-264" id="p-264"
[0264] Nucleophil groupsic on antibodies include ,but are not limite dto: (i) N-terminal amine groups, (ii) side chain amine groups, e.g. lysine, (iii) side chain thiol groups, e.g. cysteine, and (iv) sugar hydroxyl or amino groups where the antibody is glycosylated. Amine, thiol, and hydroxyl groups are nucleophilic and capable of reacting to form covalent bonds with electrophili groupsc on linke moir eties and linke reagentr s including: (i) active esters such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl halide s such as haloacetamides; and (iii) aldehydes, ketones, carboxyl ,and maleimide groups.
Certain antibodies have reducible interchain disulfide s,i.e. cysteine bridges. Antibodies may be made reactive for conjugation with linke reagentr s by treatment with a reducing agent such as DTT (dithiothreitol) or tricarbonylethylphosphine (TCEP), such that the antibody is fully or partially reduced. Each cysteine bridge wil lthus form, theoretically, two reactive thiol nucleophiles. Additional nucleophili groupsc can be introduced into antibodies through modification of lysin eresidues, e.g., by reacting lysin eresidues with 2-iminothiolane (Traut's reagent), resulting in conversion of an amine into a thiol. Reactive thiol groups may also be introduced into an antibody by introducing one, two, three, four, or more cysteine residues (e.g., by preparing variant antibodies comprising one or more non-native cysteine amino acid residues). 265. 265. id="p-265" id="p-265" id="p-265" id="p-265" id="p-265" id="p-265" id="p-265" id="p-265" id="p-265" id="p-265" id="p-265" id="p-265"
[0265] Antibody-drug conjugates of the invention may also be produced by reaction between an electrophili groupc on an antibody or antibody fragment, such as an aldehyde or ketone carbonyl group, with a nucleophili groupc on a linker reagent or drug. Useful nucleophili c 70WO 2020/092155 PCT/US2019/058066 groups on a linke reager nt include ,but are not limite dto, hydrazide ,oxime, amino, hydrazine ,thiosemicarbazone, hydrazine carboxylate, and arylhydrazide. In one embodiment, an antibody is modified to introduce electrophili moic eties that are capable of reacting with nucleophili substic tuent son the linke reager nt or drug. In another embodiment ,the sugars of glycosylated antibodies may be oxidized, e.g. with periodate oxidizing reagents, to form aldehyde or ketone groups which may react with the amine group of linke reagentr s or drug moieties The. resulting imine Schiff base groups may form a stabl elinkage, or may be reduced, e.g. by borohydride reagents to form stabl eamine linkages In. one embodiment, reaction of the carbohydrate portion of a glycosylated antibody with either galactos eoxidase or sodium meta-periodat emay yield carbonyl (aldehyde and ketone) groups in the antibody that can react with appropriate groups on the drug (Hermanson, Bioconjugate Techniques) In. another embodiment, antibodies containing N-terminal serine or threonine residue scan react with sodium meta-periodate, resulting in production of an aldehyde in place of the first amino acid (Geoghegan & Stroh, Bioconjugate Chem., vol. 3, pp. 138-146, 1992; U.S. Pat. No. ,362,852). Such an aldehyde can be reacted with a drug moiety or linke nucleophilr e. 266. 266. id="p-266" id="p-266" id="p-266" id="p-266" id="p-266" id="p-266" id="p-266" id="p-266" id="p-266" id="p-266" id="p-266" id="p-266"
[0266] Exemplary nucleophilic groups on a drug moiety include ,but are not limite dto: amine, thiol, hydroxyl, hydrazide, oxime, hydrazine ,thiosemicarbazon e,hydrazine carboxylate, and arylhydrazid egroups capable of reacting to form covalent bonds with electrophili groupsc on linker moieties and linke reagentr s including: (i) active esters such as NHS esters, HOBt esters, haloformates, and acid halide s;(ii) alkyl and benzyl halides such as haloacetamides; (iii) aldehydes, ketones carboxyl,, and maleimide groups. 267. 267. id="p-267" id="p-267" id="p-267" id="p-267" id="p-267" id="p-267" id="p-267" id="p-267" id="p-267" id="p-267" id="p-267" id="p-267"
[0267] Nonlimiting exemplary cross-linker reagents that may be used to prepare ADCs are described herei nin the section titled "Exemplary Linkers." Methods of using such cross- linke reagentr s to link two moieties incl, uding a proteinaceous moiety and a chemica lmoiety, are known in the art. In some embodiments, a fusion protein comprising an antibody and a cytotoxic agent may be made, e.g., by recombinant techniques or peptide synthesi s.A recombinan tDNA molecule may comprise regions encoding the antibody and cytotoxic portions of the conjugate either adjacent to one another or separated by a region encoding a linke peptir de which does not destroy the desired properties of the conjugate. 268. 268. id="p-268" id="p-268" id="p-268" id="p-268" id="p-268" id="p-268" id="p-268" id="p-268" id="p-268" id="p-268" id="p-268" id="p-268"
[0268] In yet another embodiment, an antibody or antibody fragment may be conjugated to a "receptor" (such as streptavidin) for utilization in tumor pre-targeting wherein the antibody/antibody fragment-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then 71WO 2020/092155 PCT/US2019/058066 administration of a "ligand" (e.g., avidin) which is conjugated to a cytotoxic agent (e.g., a drug or radionucleotide).
C. Methods and Compositions for Diagnostics and Detection 269. 269. id="p-269" id="p-269" id="p-269" id="p-269" id="p-269" id="p-269" id="p-269" id="p-269" id="p-269" id="p-269" id="p-269" id="p-269"
[0269] In certain embodiments, any of the anti-CTLA4 antibodies or antibody fragments provided herein may be used for detecting the presence of CTLA4 in a biological sample.
The term "detecting" as used herei nencompasses quantitative or qualitative detection. In certain embodiments, a biologica lsample comprises a cell or tissue, such as breast, pancreas, esophagus lung, and/or brain cells or tissue. 270. 270. id="p-270" id="p-270" id="p-270" id="p-270" id="p-270" id="p-270" id="p-270" id="p-270" id="p-270" id="p-270" id="p-270" id="p-270"
[0270] A further aspect of the invention relates to an anti-CTLA4 antibody or antibody fragment of the invention for diagnosing and/or monitoring a cancer or another diseas ein which CTLA4 expression levels are increased or decreased from a normal physiological level at at leas onet location in the body. 271. 271. id="p-271" id="p-271" id="p-271" id="p-271" id="p-271" id="p-271" id="p-271" id="p-271" id="p-271" id="p-271" id="p-271" id="p-271"
[0271] In a preferre dembodiment ,antibodies or antibody fragments of the invention may be labelle withd a detectabl emolecule or substance, such as a fluorescent molecule, a radioactive molecule or any other label known in the art as above described. For example, an antibody or antibody fragment of the invention may be labell edwith a radioactive molecul e.
For example, suitable radioactive molecules include but are not limited to radioactive atoms used for scintigraphic studies such as 123I, 124I, 111In, 186Re, and 188Re. Antibodies or antibody fragments of the invention may also be labell edwith a spin label for nuclear magnetic resonance (NMR) imaging, such as iodine-123, iodine-131, indium-Ill, fluorine-19, carbon- 13, nitrogen-15, oxygen-17, gadolinium, manganese or iron. Following administration of the antibody, the distribution of the radiolabeled antibody within the patient is detected. Any suitable known method can be used. Some non-limiting examples include ,computed tomography (CT), position emission tomography (PET), magnetic resonance imaging (MRI), fluorescence, chemiluminescence and sonography. 272. 272. id="p-272" id="p-272" id="p-272" id="p-272" id="p-272" id="p-272" id="p-272" id="p-272" id="p-272" id="p-272" id="p-272" id="p-272"
[0272] Antibodies or antibody fragments of the invention may be useful for diagnosing and staging of cancer and diseases associated with CTLA4 overexpression. Cancers associated with CTLA4 overexpression may include squamous cell cancer, small-cell lung cancer, non- smal celll lung cancer, gastric cancer, pancreatic cancer, glia lcel ltumors such as glioblastoma and neurofibromatosis, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, melanoma, colorectal cancer, endometrial carcinoma, salivary gland carcinoma, kidney cancer, renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, sarcomas, hematological cancers (leukemias), 72WO 2020/092155 PCT/US2019/058066 astrocytomas, and various types of head and neck cancer or other CTLA4 expressing or overexpressing hyperproliferative diseases. 273. 273. id="p-273" id="p-273" id="p-273" id="p-273" id="p-273" id="p-273" id="p-273" id="p-273" id="p-273" id="p-273" id="p-273" id="p-273"
[0273] Antibodies or antibody fragments of the invention may be useful for diagnosing diseases other than cancers for which CTLA4 expression is increased or decreased. Both the (soluble or cellula CTLA4r forms can be used for such diagnoses. Typically, such diagnostic methods involve use of a biological sample obtained from the patient. The biologica lsampl e encompasses a variety of sample types obtained from a subjec tthat can be used in a diagnostic or monitoring assay. Biological samples include but are not limite dto blood and other liquid samples of biologica lorigin, soli dtissue sample suchs as a biopsy specimen or a tissue culture or cells derived therefrom and, the progeny thereof. For example, biological samples include cell obtais ned from a tissue sample collected from an individual suspected of having a cancer associated with CTLA4 overexpressio n,and in preferred embodiments from glioma, gastric, lung ,pancreatic, breast ,prostate, renal ,hepatic and endometrial. Biologica l samples encompass clinical samples, cells in culture, cel lsupernatants, cel llysates, serum, plasma, biological fluid, and tissue samples. 274. 274. id="p-274" id="p-274" id="p-274" id="p-274" id="p-274" id="p-274" id="p-274" id="p-274" id="p-274" id="p-274" id="p-274" id="p-274"
[0274] In a particular embodiment ,the invention is a method of diagnosing a cancer associated with CTLA4 overexpression in a subject by detecting CTLA4 on cells from the subject using the antibody of the invention. In particular, said method may include steps of: (a) contacting a biological sample of a subject with an antibody or antibody fragment according to the invention under conditions suitable for the antibody or antibody fragment to form complexes with cells in the biological sampl ethat express CTLA4; and (b) detecting and/or quantifying said complexes whereby, detection of said complexes is indicative of a cancer associated with CTLA4 overexpression. 275. 275. id="p-275" id="p-275" id="p-275" id="p-275" id="p-275" id="p-275" id="p-275" id="p-275" id="p-275" id="p-275" id="p-275" id="p-275"
[0275] In order to monitor the progress of a cancer, the method according to the invention may be repeated at different times, in order to determine if antibody binding to the samples increases or decreases, wherefrom it can be determined if the cancer has progressed , regressed or stabilized. 276. 276. id="p-276" id="p-276" id="p-276" id="p-276" id="p-276" id="p-276" id="p-276" id="p-276" id="p-276" id="p-276" id="p-276" id="p-276"
[0276] In a particular embodiment ,the invention is a method of diagnosing a disease associated with the expression or overexpression of CTLA4 or a decrease or increase of the solubl forme of CTLA4. Examples of such diseases may include human immune disorders, thrombotic diseases (thrombosi sand atherothrombosis) and, cardiovascular diseases 277. 277. id="p-277" id="p-277" id="p-277" id="p-277" id="p-277" id="p-277" id="p-277" id="p-277" id="p-277" id="p-277" id="p-277" id="p-277"
[0277] In one embodiment ,an anti-CTLA4 antibody or antibody fragment for use in a method of diagnosis or detection is provided. In a further aspect, a method of detecting the presence of CTLA4 in a biological sample is provided. In a further aspect, a method of 73WO 2020/092155 PCT/US2019/058066 quantifying the amount of CTLA4 in a biological sample is provided. In certain embodiments, the method comprises contacting the biological sampl ewith an anti-CTLA4 antibody or antibody fragment as described herei nunder conditions permissive for binding of the anti-CTLA4 antibody or antibody fragment to CTLA4, and detecting whether a comple x is formed between the anti-CTLA4 antibody or antibody fragment and CTLA4. Such a method may be carried out in vitro or in vivo. In one embodiment ,an anti-CTLA4 antibody or antibody fragment is used to select subjects eligible for therapy. In some embodiments, the therapy will include administration of an anti-CTLA4 antibody or antibody fragment to the subject. 278. 278. id="p-278" id="p-278" id="p-278" id="p-278" id="p-278" id="p-278" id="p-278" id="p-278" id="p-278" id="p-278" id="p-278" id="p-278"
[0278] In certain embodiments, labeled anti-CTLA4 antibodies or antibody fragments are provided. Labels include ,but are not limite dto, labels or moieties that are detected directly (such as fluorescent, chromophoric, electron-dense chemi, luminesce andnt, radioactive labels), as wel las moieties, such as enzymes or ligands, that are detected indirectly, e.g., through an enzymatic reaction or molecular interaction. Exemplary label include,s but are not limite dto, the radioisotopes 32P, 14C, 1251,3H, and 131I, fluorophores such as rare earth chelates or fluorescei andn its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luceriferases e.g.,, firefly luciferase and bacterial luciferase (U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones horse, radish peroxidase (HRP), alkaline phosphatas e,P־galactosidase, glucoamylas e,lysozyme, saccharide oxidases, e.g., glucose oxidase, galactos eoxidase ,and glucose-6-phosphat dehydrogenasee heteroc, yclic oxidases such as uricase and xanthine oxidase, coupled with an enzyme that employs hydrogen peroxide to oxidize a dye precursor such as HRP, lactoperoxidase, or microperoxidase, biotin/avidin, spin labels, bacteriophage labels stabl, efree radicals ,and the like.
D. Pharmaceutical Formulations 279. 279. id="p-279" id="p-279" id="p-279" id="p-279" id="p-279" id="p-279" id="p-279" id="p-279" id="p-279" id="p-279" id="p-279" id="p-279"
[0279] The anti-CTLA4 antibodies or antibody fragments have cell killing activity. This cel l killing activity extends to multiple different types of cel llines. Further, these antibodies or antibody fragments ,once conjugated to a cytotoxic agent, can reduce tumor size and may exhibit reduced toxicity. Thus, the anti-CTLA4 antibodies ,fragments or immunoconjugates thereof may be useful for treating proliferative diseases associated with CTLA4 expression.
The antibodies ,fragments or immunoconjugates may be used alone or in combination with any suitable agent or other conventional treatments. 280. 280. id="p-280" id="p-280" id="p-280" id="p-280" id="p-280" id="p-280" id="p-280" id="p-280" id="p-280" id="p-280" id="p-280" id="p-280"
[0280] The anti-CTLA4 antibody or antibody fragment may be used to treat diseases associated with CTLA4 expression, overexpression or activation. There are no particular 74WO 2020/092155 PCT/US2019/058066 limitations on the types of cancer or tissue that can be treated other than the requirement for CTLA4 expression. Examples include squamous cell cancer, small-ce lungll cancer, non- smal celll lung cancer, gastric cancer, pancreatic cancer, glia lcel ltumors such as glioblastoma and neurofibromatosis, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, melanoma, colorectal cancer, endometrial carcinoma, salivary gland carcinoma, kidney cancer, renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, sarcomas, hematological cancers (leukemias), astrocytomas, and various types of head and neck cancer. More preferabl ecancers are glioma, gastric, lung ,pancreatic, breast ,prostate, renal ,hepatic and endometria lcancer. 281. 281. id="p-281" id="p-281" id="p-281" id="p-281" id="p-281" id="p-281" id="p-281" id="p-281" id="p-281" id="p-281" id="p-281" id="p-281"
[0281] Anti-CTLA4 antibodies or antibody fragments are potential activators of the innate immune response and thus may be used in the treatment of human immune disorders, such as sepsis .The anti-CTLA4 antibody or antibody fragment of the invention may also be used as adjuvants for immunization such as for vaccines and as anti-infection agents against, for example, bacteria, viruses and parasites. 282. 282. id="p-282" id="p-282" id="p-282" id="p-282" id="p-282" id="p-282" id="p-282" id="p-282" id="p-282" id="p-282" id="p-282" id="p-282"
[0282] Anti-CTLA4 antibody or antibody fragment may be used to protect against, prevent or treat thrombotic diseases such as venous and arterial thrombosis and atherothrombosis Anti. - CTLA4 antibody or antibody fragment may also be used to protect against, prevent or treat cardiovascular diseases as wel las to preven tor inhibit the entry of viruses such as Lassa and Ebola viruses and to treat viral infections. 283. 283. id="p-283" id="p-283" id="p-283" id="p-283" id="p-283" id="p-283" id="p-283" id="p-283" id="p-283" id="p-283" id="p-283" id="p-283"
[0283] In each of the embodiments of the treatment methods described herein, the anti- CTLA4 antibody, antibody fragment or anti-CTLA4 antibody or antibody fragment immunoconjugate may be delivered in a manner consistent with conventional methodologies associated with management of the diseas eor disorder for which treatment is sought. In accordance with the disclosure herein, an effective amount of the antibody, antibody fragment or immunoconjugate is administered to a subjec tin need of such treatment for a time and under conditions sufficient to preven tor treat the diseas eor disorder. Thus, an aspect of the invention relates to a method for treating a diseas eassociated with the expression of CTLA4 comprising administering to a subjec tin need thereof with a therapeutical lyeffective amount of an antibody, antibody fragment or immunoconjugate of the invention. 284. 284. id="p-284" id="p-284" id="p-284" id="p-284" id="p-284" id="p-284" id="p-284" id="p-284" id="p-284" id="p-284" id="p-284" id="p-284"
[0284] For administration, the anti-CTLA4 antibody, antibody fragment or immunoconjugate may be formulated as a pharmaceutical composition. The pharmaceutical composition including anti-CTLA4 antibody, antibody fragment or immunoconjugate can be formulated according to known methods for preparing pharmaceutical compositions .In such methods, 75WO 2020/092155 PCT/US2019/058066 the therapeuti cmolecul ise typically combined with a mixture, solution or composition containing a pharmaceutical lyacceptable carrier. 285. 285. id="p-285" id="p-285" id="p-285" id="p-285" id="p-285" id="p-285" id="p-285" id="p-285" id="p-285" id="p-285" id="p-285" id="p-285"
[0285] A pharmaceuticall acceptabley carrier is a materia lthat can be tolerate dby a recipient patient. Sterile phosphate-buffered saline is one example of a pharmaceutical lyacceptable carrier. Other suitable pharmaceutical lyacceptable carriers are well-known to those in the art.
(See, e.g., Gennaro (ed.), Remington's Pharmaceutical Sciences (Mack Publishing Company, 19th ed. 1995)) Formulations may further include one or more excipients, preservatives, solubilizers, buffering agents, albumin to preven tprotein los son vial surfaces, etc. 286. 286. id="p-286" id="p-286" id="p-286" id="p-286" id="p-286" id="p-286" id="p-286" id="p-286" id="p-286" id="p-286" id="p-286" id="p-286"
[0286] The form of the pharmaceutica lcompositions ,the route of administration, the dosage and the regimen naturally depend upon the condition to be treated, the severity of the illness, the age, weight, and sex of the patient, etc. These considerations can be taken into account by a skilled person to formulate suitable pharmaceutical compositions . The pharmaceutical compositions of the invention can be formulated for topical, oral, parenteral intranasal, , intravenous, intramuscular, subcutaneous or intraocula radministration and the like. 287. 287. id="p-287" id="p-287" id="p-287" id="p-287" id="p-287" id="p-287" id="p-287" id="p-287" id="p-287" id="p-287" id="p-287" id="p-287"
[0287] Preferably, the pharmaceutical compositions contain vehicles which are pharmaceuticall accy eptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium , potassium ,calcium or magnesium chloride and the like or mixtures of such salts), or dry, especiall freezy e-dried compositions which upon addition of, for example, sterilized water or physiological saline, permit the constitution of injectable solutions. 288. 288. id="p-288" id="p-288" id="p-288" id="p-288" id="p-288" id="p-288" id="p-288" id="p-288" id="p-288" id="p-288" id="p-288" id="p-288"
[0288] In some embodiments tonici, ty agents ,sometimes known as "stabilizers" are present to adjust or maintain the tonicity of a liquid in a composition. When used with large, charged biomolecul essuch as proteins and antibodies ,they are often termed "stabilizers" because they can interact with the charged groups of the amino acid side chains, thereby lessening the potential for inter- and intra-molecula intr eractions. Tonicity agents can be present in any amount of from 0.1% to 25% by weight ,preferably 1 to 5% of the pharmaceutical composition. Preferred tonicity agents include polyhydric sugar alcohol s,preferably trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol and mannitol . 289. 289. id="p-289" id="p-289" id="p-289" id="p-289" id="p-289" id="p-289" id="p-289" id="p-289" id="p-289" id="p-289" id="p-289" id="p-289"
[0289] Additional excipients include agents which can serve as one or more of the following: (1) bulking agents, (2) solubili tyenhancers, (3) stabilizers and (4) and agents preventing denaturation or adherence to the container wall. Such excipients may include :polyhydric sugar alcohol (enumerates dabove); amino acids such as alanine, glycine, glutamine , asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid, threonine, etc.; organic sugars or sugar alcohol suchs as sucrose ,lactose lactitol, trehal, ose, 76WO 2020/092155 PCT/US2019/058066 stachyose, mannose, sorbose, xylose, ribose ,ribitol ,myoinisitose, myoinisitol gala, ctose, galactitol glycerol, cycl, itols (e.g., inositol), polyethylene glycol; sulfur containing reducing agents ,such as urea, glutathione, thioctic acid, sodium thioglycolat e,thioglycerol, a- monothioglycerol and sodium thio sulfate; low molecula weir ght proteins such as human serum albumin, bovine serum albumin, gelatin or other immunoglobulins; hydrophili c polymers such as polyvinylpyrrolidone; monosaccharides (e.g., xylose, mannose ,fructose, glucose; disaccharide s(e.g., lactose mal, tose sucr, ose) ;trisaccharides such as raffinose ;and polysaccharides such as dextrin or dextran. 290. 290. id="p-290" id="p-290" id="p-290" id="p-290" id="p-290" id="p-290" id="p-290" id="p-290" id="p-290" id="p-290" id="p-290" id="p-290"
[0290] Non-ionic surfactants or detergents (also known as "wetting agents") may be employed to help solubilize the therapeutic agent as wel las to protect the therapeuti cprotein against agitation-induced aggregation, which also permits the formulation to be exposed to shear surface stress without causing denaturation of the active therapeutic protein or antibody. Non-ionic surfactants may be present in a concentration range of about 0.05 mg/ml to about 1.0 mg/ml, preferably about 0.07 mg/ml to about 0.2 mg/ml. 291. 291. id="p-291" id="p-291" id="p-291" id="p-291" id="p-291" id="p-291" id="p-291" id="p-291" id="p-291" id="p-291" id="p-291" id="p-291"
[0291] Suitable non-ionic surfactants include polysorbate s(20, 40, 60, 65, 80, etc.), polyoxamers (184, 188, etc.), PLURONIC® polyols, TRITON®, polyoxyethylene sorbitan monoethers (TWEEN®-20, TWEEN®-80, etc.), lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate sucros, e fatty acid ester, methyl celluose and carboxymethyl cellulose. Anionic detergents that can be used include sodium lauryl sulfate, dioctyle sodium sulfosuccinate and dioctyl sodium sulfonate.
Cationic detergents include benzalkonium chloride or benzethonium chloride 292. 292. id="p-292" id="p-292" id="p-292" id="p-292" id="p-292" id="p-292" id="p-292" id="p-292" id="p-292" id="p-292" id="p-292" id="p-292"
[0292] The doses used for the administration can be adapted as a function of various parameters, and in particular as a function of the mode of administration used, of the relevant pathology, or alternatively of the desired duration of treatment .To prepare pharmaceutical compositions ,an effective amount of the antibody or antibody fragment may be dissolved or dispersed in a pharmaceuticall accepty able carrier or aqueous medium. 293. 293. id="p-293" id="p-293" id="p-293" id="p-293" id="p-293" id="p-293" id="p-293" id="p-293" id="p-293" id="p-293" id="p-293" id="p-293"
[0293] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stabl eunder the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. 77WO 2020/092155 PCT/US2019/058066 294. 294. id="p-294" id="p-294" id="p-294" id="p-294" id="p-294" id="p-294" id="p-294" id="p-294" id="p-294" id="p-294" id="p-294" id="p-294"
[0294] Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in a water suitably mixed with a surfactant. Dispersions can also be prepared in glycerol liqui, d polyethylene glycols, and mixtures thereof and in oils .Under ordinary conditions of storage and use, thes epreparations contain a preservative to prevent the growth of microorganisms. 295. 295. id="p-295" id="p-295" id="p-295" id="p-295" id="p-295" id="p-295" id="p-295" id="p-295" id="p-295" id="p-295" id="p-295" id="p-295"
[0295] The anti-CTLA4 antibody or antibody fragment can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salt sinclude the acid addition salt s (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochlor icor phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium ,calcium, or ferric hydroxides, and such organic bases as isopropylamine tri, methylamine, histidine, procaine and the like. 296. 296. id="p-296" id="p-296" id="p-296" id="p-296" id="p-296" id="p-296" id="p-296" id="p-296" id="p-296" id="p-296" id="p-296" id="p-296"
[0296] The carrier can also be a solvent or dispersio nmedium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils .The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersio nand by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents ,for example, parabens, chlorobutanol phenol,, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents ,for example, sugars or sodium chloride. Prolonged absorption of the injectabl ecompositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. 297. 297. id="p-297" id="p-297" id="p-297" id="p-297" id="p-297" id="p-297" id="p-297" id="p-297" id="p-297" id="p-297" id="p-297" id="p-297"
[0297] Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with one or more of the other ingredients enumerated above, as may be required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersio nmedium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of steril e injectable solutions, the preferred methods of preparation are vacuum-drying and freeze- drying technique swhich yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. 78WO 2020/092155 PCT/US2019/058066 298. 298. id="p-298" id="p-298" id="p-298" id="p-298" id="p-298" id="p-298" id="p-298" id="p-298" id="p-298" id="p-298" id="p-298" id="p-298"
[0298] The preparation of more, or highly concentrated solutions for direct injection is also contemplated, where the use of dimethyl sulfoxide (DMSO) as solvent is envisioned to resul t in extremel rapidy penetration, delivering high concentrations of the active agents to a smal l tumor area. 299. 299. id="p-299" id="p-299" id="p-299" id="p-299" id="p-299" id="p-299" id="p-299" id="p-299" id="p-299" id="p-299" id="p-299" id="p-299"
[0299] Upon formulation, solutions wil lbe administered in a manner compatible with the dosage formulation and in such amount as is therapeuticall effecy tive .The formulations are easil yadministered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed. 300. 300. id="p-300" id="p-300" id="p-300" id="p-300" id="p-300" id="p-300" id="p-300" id="p-300" id="p-300" id="p-300" id="p-300" id="p-300"
[0300] For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendere disotonic with sufficient saline or glucose. These particular aqueous solutions are especial lysuitable for intravenous, intramuscular subcut, aneous and intraperitonea ladministration. In this connection, sterile aqueous media which can be employed wil lbe known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysi fluis d or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subjec tbeing treated. The person responsibl fore administration will, in any event, determine the appropriate dose for the individual subject. 301. 301. id="p-301" id="p-301" id="p-301" id="p-301" id="p-301" id="p-301" id="p-301" id="p-301" id="p-301" id="p-301" id="p-301" id="p-301"
[0301] The antibodies or antibody fragments may be formulated within a therapeuti cmixture to deliver about 0.0001 to 10.0 milligrams, or about 0.001 to 5 milligrams, or about 0.001 to 1 milligrams, or about 0.001 to 0.1 milligrams, or about 0.1 to 1.0 or even about 10 milligrams per dose. Multiple doses can also be administered at selected time intervals. 302. 302. id="p-302" id="p-302" id="p-302" id="p-302" id="p-302" id="p-302" id="p-302" id="p-302" id="p-302" id="p-302" id="p-302" id="p-302"
[0302] In addition to the compounds formulated for parenteral administration, such as intravenous or intramuscular injection, other pharmaceutical lyacceptable forms include, e.g. tablets or other solids for oral administration; time releas capse ules and; any other form currentl yused. 303. 303. id="p-303" id="p-303" id="p-303" id="p-303" id="p-303" id="p-303" id="p-303" id="p-303" id="p-303" id="p-303" id="p-303" id="p-303"
[0303] In certain embodiments, the use of liposomes and/or nanoparticle sis contemplate ford the introduction of antibodies or antibody fragments into host cells. The formation and use of liposomes and/or nanoparticles are known to those of skill in the art. 304. 304. id="p-304" id="p-304" id="p-304" id="p-304" id="p-304" id="p-304" id="p-304" id="p-304" id="p-304" id="p-304" id="p-304" id="p-304"
[0304] Nanocapsules can generally entrap compounds in a stable and reproducible way. To avoid side effects due to intracellul polymeriar coverloading, such ultrafine particles (sized around 0.1 pm) are generally designed using polymers able to degrade in vivo. Biodegradabl e 79WO 2020/092155 PCT/US2019/058066 polyalkyl-cyanoacryl nanoparticlate es that meet these requirements are contemplate ford use in the present invention, and such particle smay be easil ymade. 305. 305. id="p-305" id="p-305" id="p-305" id="p-305" id="p-305" id="p-305" id="p-305" id="p-305" id="p-305" id="p-305" id="p-305" id="p-305"
[0305] Liposomes are formed from phospholipi dsthat are dispersed in an aqueous medium and spontaneously form multilamell concear ntric bilayer vesicle (als so termed multilamel lar vesicles (MLVs)). MLVs generally have diameters of from 25 nm to 4 pm. Sonication of MLVs result sin the formation of smal unilaml ell vesicar les (SUVs) with diameters in the range of 200 to 500 A, containing an aqueous solution in the core. The physical characteristics of liposomes depend on pH, ionic strength and the presence of divalent cations 306. 306. id="p-306" id="p-306" id="p-306" id="p-306" id="p-306" id="p-306" id="p-306" id="p-306" id="p-306" id="p-306" id="p-306" id="p-306"
[0306] Pharmaceutical formulations containing an anti-CTLA4 antibody or antibody fragment as described herei nare prepared by mixing such antibody or antibody fragment having the desired degree of purity with one or more optional pharmaceutical lyacceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limite dto: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol );low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin ,gelatin, or immunoglobulins hydrophilic; polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine ,histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose ,or dextrins; chelating agents such as EDTA; sugars such as sucrose ,mannitol ,trehalos ore sorbitol; salt-forming counter-ions such as sodium ;meta lcomplexe (e.g.s Zn-protein complexes); and/or non-ionic surfactants such as polyethylene glycol (PEG). 307. 307. id="p-307" id="p-307" id="p-307" id="p-307" id="p-307" id="p-307" id="p-307" id="p-307" id="p-307" id="p-307" id="p-307" id="p-307"
[0307] Exemplary pharmaceutical lyacceptable carriers herei nfurther include insterstitial drug dispersion agents such as solubl neutral-actie ve hyaluronidase glycoproteins (sHASEGP), for example, human solubl PH-20e hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International ,Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in US Patent Publication Nos. 2005/0260186 and 2006/0104968. In one aspect, a sHASEGP is combined with one or more additional glycosaminoglycanases such as chondroitinases. 80WO 2020/092155 PCT/US2019/058066 308. 308. id="p-308" id="p-308" id="p-308" id="p-308" id="p-308" id="p-308" id="p-308" id="p-308" id="p-308" id="p-308" id="p-308" id="p-308"
[0308] Exemplary lyophilized antibody formulations are described in U.S. Patent No. 6,267,958. Aqueous antibody formulations include those described in U.S. Patent No. 6,171,586 and WO2006/044908, the latte rformulations including a histidine-acetat buffer.e 309. 309. id="p-309" id="p-309" id="p-309" id="p-309" id="p-309" id="p-309" id="p-309" id="p-309" id="p-309" id="p-309" id="p-309" id="p-309"
[0309] The formulation herein may also contain more than one active ingredient as necessary for the particular indication being treated. Preferably, ingredients with complementar y activities that do not adversely affect each other may be combined into a singl eformulation.
For example, it may be desirable to provide an EGFR antagonist (such as erlotinib) ,an anti- angiogenic agent (such as a VEGF antagonist which may be an anti-VEGF antibody) or a chemotherapeuti agentc (such as a taxoid or a platinum agent) in addition to the anti-CTLA4 antibody, antibody fragment or immunoconjugate of the present invention. Such active ingredients are suitably present in combination in amounts that are effective for the purpose intended. 310. 310. id="p-310" id="p-310" id="p-310" id="p-310" id="p-310" id="p-310" id="p-310" id="p-310" id="p-310" id="p-310" id="p-310" id="p-310"
[0310] In one embodiment the, anti-CTLA4 antibody, antibody fragment or immunoconjugate of the present invention is combined in a formulation with another antibody or antibody fragment against an antigen selected from PD1, PD-L1, AXL, ROR2, CD3, HER2, B7-H3, ROR1, SFRP4 and a WNT protein including WNT1, WNT2, WNT2B, WNT3, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, WNT16. The combination may be in the form of two separate molecules: the anti-CTLA4 antibody, antibody fragment or immunoconjugate of the present invention, and the another antibody or antibody fragment. Alternatively the, combination may also be the form of a singl emolecul wite h binding affinity to both CTLA4 and the other antigen, thus forming a multispecif ic(e.g. bispecific) antibody. 311. 311. id="p-311" id="p-311" id="p-311" id="p-311" id="p-311" id="p-311" id="p-311" id="p-311" id="p-311" id="p-311" id="p-311" id="p-311"
[0311] Active ingredients may be encapsulated in microcapsules prepared, for example, by coacervation technique sor by interfacial polymerization. For example, hydroxymethylcellu orlose gelatin-microcapsules and poly-(methylmethacyla te) microcapsules, respectively, in colloidal drug deliver ysystems (for example, liposome s, albumin microspheres mi, croemulsions, nano-particle sand nanocapsules or) in macroemulsions may be employed. Such technique sare disclose ind Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). 312. 312. id="p-312" id="p-312" id="p-312" id="p-312" id="p-312" id="p-312" id="p-312" id="p-312" id="p-312" id="p-312" id="p-312" id="p-312"
[0312] Sustained-release preparations may be prepared. Suitable examples of sustained- releas preparate ions include semipermeable matrices of soli dhydrophobic polymers containing the antibody or antibody fragment, which matrices may be in the form of shaped articles, e.g. films, or microcapsules. 313. 313. id="p-313" id="p-313" id="p-313" id="p-313" id="p-313" id="p-313" id="p-313" id="p-313" id="p-313" id="p-313" id="p-313" id="p-313"
[0313] The formulations to be used for in vivo administration are generally sterile. Sterility 81WO 2020/092155 PCT/US2019/058066 may be readily accomplished, e.g., by filtration through sterile filtration membranes.
E. Therapeutic Methods and Compositions 314. 314. id="p-314" id="p-314" id="p-314" id="p-314" id="p-314" id="p-314" id="p-314" id="p-314" id="p-314" id="p-314" id="p-314" id="p-314"
[0314] Any of the anti-CTLA4 antibodies or antibody fragments provided herei nmay be used in therapeutic methods In. one aspect, an anti-CTLA4 antibody or antibody fragment for use as a medicament is provided. In further aspects, an anti-CTLA4 antibody or antibody fragment for use in treating cancer (e.g., breast cancer, non-small cel llung cancer, pancreatic cancer, brain cancer, cancer of pancreas, brain, kidney, ovary, stomach, leukemi a,uterine endometrium, colon, prostate, thyroid, liver, osteosarcoma and/or, melanoma) is provided. In certain embodiments, an anti-CTLA4 antibody or antibody fragment for use in a method of treatment is provided. In certain embodiments, the invention provides an anti-CTLA4 antibody or antibody fragment for use in a method of treating an individual having cancer comprising administering to the individual an effective amount of the anti-CTLA4 antibody or antibody fragment. In certain embodiments, the invention provides an anti-CTLA4 antibody or antibody fragment for use in a method of treating an individual having an immune disorder (e.g., an autoimmune disorder), a cardiovascular disorder (e.g., atheroscleros hypertensiis, on, thrombosis), an infectious diseas e(e.g., Ebola virus, Marburg virus) or diabetes ,comprising administering to the individual an effective amount of the anti- CTLA4 antibody or antibody fragment .In one such embodiment the, method further comprises administering to the individual an effective amount of at least one additional therapeuti cagent, e.g., as described below. In further embodiments the, invention provides an anti-CTLA4 antibody or antibody fragment for use in inhibiting angiogenesis, inhibiting cell proliferation, inhibiting immune function, inhibiting inflammatory cytokine secretion (e.g., from tumor-associated macrophages), inhibiting tumor vasculature (e.g., intratumoral vasculature or tumor-associated vasculature), and/or inhibiting tumor stromal function. 315. 315. id="p-315" id="p-315" id="p-315" id="p-315" id="p-315" id="p-315" id="p-315" id="p-315" id="p-315" id="p-315" id="p-315" id="p-315"
[0315] In certain embodiments, the invention provides an anti-CTLA4 antibody or antibody fragment for use in a method of inhibiting angiogenesis, inhibiting cell proliferation, inhibiting immune function, inhibiting inflammatory cytokine secretion (e.g., from tumor- associated macrophages), inhibiting tumor vasculature (e.g., intratumoral vasculature or tumor-associated vasculature ),and/or inhibiting tumor stromal function in an individual comprising administering to the individual an effective amount of the anti-CTLA4 antibody or antibody fragment to inhibit angiogenesis, inhibit cell proliferation, inhibit immune function, inhibit inflammator ycytokine secretion (e.g., from tumor-associated macrophages), inhibit tumor vasculature development (e.g., intratumoral vasculature or tumor-associate d 82WO 2020/092155 PCT/US2019/058066 vasculature), and/or inhibit tumor stromal function. An "individual" according to any of the above embodiments is preferabl ya human. 316. 316. id="p-316" id="p-316" id="p-316" id="p-316" id="p-316" id="p-316" id="p-316" id="p-316" id="p-316" id="p-316" id="p-316" id="p-316"
[0316] In a further aspect, the invention provides for the use of an anti-CTLA4 antibody or antibody fragment in the manufacture or preparation of a medicamen t.In one embodiment, the medicament is for treatment of cancer (in some embodiments, breast cancer, non-small cell lung cancer, pancreatic cancer, brain cancer, cancer of the pancreas ,brain, kidney, ovary, stomach, leukem ia,uterine endometrium, colon ,prostate, thyroid, liver, osteosarcoma and/o, r melanoma) In. a further embodiment ,the medicament is for use in a method of treating cancer comprising administering to an individual having cancer an effective amount of the medicament. In a further embodiment ,the medicament is for use in a method of treating an immune disorder (e.g., an autoimmune disorder), a cardiovascular disorder (e.g., atheroscleros hypertensiis, on, thrombosis), an infectious diseas e(e.g., Ebola virus, Marburg virus) or diabetes ,comprising administering to the individual an effective amount of the anti- CTLA4 antibody or antibody fragment .In one such embodiment the, method further comprises administering to the individual an effective amount of at least one additional therapeuti cagent, e.g., as described below. In a further embodiment ,the medicament is for inhibiting angiogenesis, inhibiting cel lproliferation, inhibiting immune function, inhibiting inflammatory cytokine secretion (e.g., from tumor-associated macrophages), inhibiting tumor vasculature (e.g., intratumoral vasculatur eor tumor-associated vasculature), and/or inhibiting tumor stromal function. In a further embodiment ,the medicamen ist for use in a method of inhibiting angiogenesis, inhibiting cel lproliferation, inhibiting immune function, inhibiting inflammatory cytokine secretion (e.g., from tumor-associated macrophages), inhibiting tumor vasculature (e.g., intratumoral vasculatur eor tumor-associated vasculature), and/or inhibiting tumor stromal function in an individual comprising administering to the individual an amount effective of the medicament to inhibit angiogenesis inhi, bit cell proliferation, promote immune function, induce inflammatory cytokine section (e.g., from tumor-associated macrophages), inhibit tumor vasculature developme nt(e.g., intratumoral vasculatur eor tumor-associated vasculature ),and/or inhibit tumor stromal function. An "individual" according to any of the above embodiments may be a human. 317. 317. id="p-317" id="p-317" id="p-317" id="p-317" id="p-317" id="p-317" id="p-317" id="p-317" id="p-317" id="p-317" id="p-317" id="p-317"
[0317] In a further aspect, the invention provides a method for treating a cancer. In one embodiment the, method comprises administering to an individual having such cancer an effective amount of an anti-CTLA4 antibody or antibody fragment .In one such embodiment, the method further comprises administering to the individual an effective amount of at least 83WO 2020/092155 PCT/US2019/058066 one additional therapeuti cagent, as described below. An "individual" according to any of the above embodiments may be a human. 318. 318. id="p-318" id="p-318" id="p-318" id="p-318" id="p-318" id="p-318" id="p-318" id="p-318" id="p-318" id="p-318" id="p-318" id="p-318"
[0318] In a further aspect, the invention provides a method for treating an immune disorder (e.g., an autoimmune disorder), a cardiovascula rdisorder (e.g., atherosclerosis, hypertension, thrombosis), an infectious diseas e(e.g., Ebola virus, Marburg virus) or diabetes. In one such embodiment the, method further comprise sadministering to the individual an effective amount of at least one additional therapeuti cagent, as described below. An "individual" according to any of the above embodiments may be a human. 319. 319. id="p-319" id="p-319" id="p-319" id="p-319" id="p-319" id="p-319" id="p-319" id="p-319" id="p-319" id="p-319" id="p-319" id="p-319"
[0319] In a further aspect, the invention provides a method for inhibiting angiogenesis, inhibiting cell proliferation, inhibiting immune function, inhibiting inflammatory cytokine secretion (e.g., from tumor-associated macrophages), inhibiting tumor vasculature (e.g., intratumoral vasculature or tumor-associated vasculature), and/or inhibiting tumor stromal function in an individual .In one embodiment, the method comprises administering to the individual an effective amount of an anti-CTLA4 antibody or antibody fragment to inhibit angiogenesis, inhibit cell proliferation, promote immune function, induce inflammator y cytokine section (e.g., from tumor-associated macrophages), inhibit tumor vasculature development (e.g., intratumoral vasculature or tumor-associated vasculature), and/or inhibit tumor stromal function. In one embodiment, an "individual" is a human. 320. 320. id="p-320" id="p-320" id="p-320" id="p-320" id="p-320" id="p-320" id="p-320" id="p-320" id="p-320" id="p-320" id="p-320" id="p-320"
[0320] In a further aspect, the invention provides pharmaceutical formulations comprising any of the anti-CTLA4 antibodies or antibody fragments provided herein, e.g., for use in any of the above therapeutic methods. In one embodiment, a pharmaceutical formulation comprises any of the anti-CTLA4 antibodies or antibody fragments provided herein and a pharmaceuticall accy eptable carrier. In another embodiment ,a pharmaceutical formulation comprises any of the anti-CTLA4 antibodies or antibody fragments provided herein and at least one additional therapeuti cagent, e.g., as described below. 321. 321. id="p-321" id="p-321" id="p-321" id="p-321" id="p-321" id="p-321" id="p-321" id="p-321" id="p-321" id="p-321" id="p-321" id="p-321"
[0321] In each and every treatment described above, the antibodies or antibody fragments of the invention can be used alone, as immunoconjugates or in combination with other agents in a therapy. For instance, an antibody of the invention may be co-administered with at least one additional therapeuti cagent. In certain embodiments, an additional therapeuti cagent is an anti-angiogenic agent. In certain embodiments, an additional therapeuti cagent is a VEGF antagonist (in some embodiments, an anti-VEGF antibody, for example bevacizumab). In certain embodiments, an additional therapeuti cagent is an EGFR antagonist (in some embodiment erlot, inib) .In certain embodiments, an additional therapeuti cagent is a chemotherapeuti agentc and/or a cytostatic agent. In certain embodiments, an additional 84WO 2020/092155 PCT/US2019/058066 therapeuti cagent is a taxoid (e.g., paclitaxel and/) or a platinum agent (e.g., carboplatinum).
In certain embodiments the additional therapeuti cagent is an agent that enhances the patient’s immunity or immune system. 322. 322. id="p-322" id="p-322" id="p-322" id="p-322" id="p-322" id="p-322" id="p-322" id="p-322" id="p-322" id="p-322" id="p-322" id="p-322"
[0322] Such combination therapies noted above encompass combined administration (where two or more therapeutic agents are included in the same or separate formulations), and separate administration, in which case ,administration of the antibody or antibody fragment can occur prior to, simultaneously, and/or following, administration of the additional therapeuti cagent and/or adjuvant. Antibodies or antibody fragments can also be used in combination with radiation therapy. 323. 323. id="p-323" id="p-323" id="p-323" id="p-323" id="p-323" id="p-323" id="p-323" id="p-323" id="p-323" id="p-323" id="p-323" id="p-323"
[0323] The anti-CTLA4 antibodies or antibody fragments may be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners .The antibody or antibody fragment need not be, but is optionall yformulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents depends on the amount of antibody or antibody fragment present in the formulation, the type of disorder or treatment, and othe r factors discussed above. These are generally used in the same dosages and with administration routes as described herein, or about from 1 to 99% of the dosages described herein, or in any dosage and by any route that is empirically/clinicall determy ined to be appropriate. 324. 324. id="p-324" id="p-324" id="p-324" id="p-324" id="p-324" id="p-324" id="p-324" id="p-324" id="p-324" id="p-324" id="p-324" id="p-324"
[0324] For the prevention or treatment of disease, the appropriate dosage of an antibody or antibody fragment (when used alone or in combination with one or more other additional therapeuti cagents) wil ldepend on the type of disease to be treated, the type of antibody or antibody fragment ,the severity and course of the disease, whether the antibody or antibody fragment is administered for preventive or therapeuti cpurposes, previous therapy, the patient's clinical history and response to the antibody or antibody fragment ,and the discretion of the attending physician. The antibody or antibody fragment is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 pg of antibody or antibody fragment /kg body weight of the patient to 40 mg of antibody or antibody fragment /kg bodyweight of the patient can be an initial candidate dosage for administration to the patient, whethe r,for example, by one or more separate administrations, or by continuous infusion. One typical daily dosage might range from about 85WO 2020/092155 PCT/US2019/058066 1 pg of antibody or antibody fragment /kg body weight of the patient to 100 mg of antibody or antibody fragment /kg bodyweight of the patient or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment would generally be sustained until a desired suppression of diseas esymptoms occurs. Such doses may be administered intermittently, e.g. every week or every three weeks (e.g. such that the patient receives from about two to about twenty, or e.g. about six doses of the antibody or antibody fragment). An initial higher loading dose, followed by one or more lower doses may be administered. However, other dosage regimens may be useful. The progress of this therapy is easil ymonitored by conventional techniques and assays. 325. 325. id="p-325" id="p-325" id="p-325" id="p-325" id="p-325" id="p-325" id="p-325" id="p-325" id="p-325" id="p-325" id="p-325" id="p-325"
[0325] Specific dosages of the anti-CTLA4 antibody or antibody fragment of the present invention that may be administered for the prevention or treatment of a diseas ein a subjec t may be about 0.3, 0.6, 1.2, 18, 2.4, 3.0, 3.6, 4.2, 4.8, 5.4, 6.0, 6.6, 7.2, 7.8, 8.4, 9.0, 9.6 or .2 mg of antibody or antibody fragment /kg bodyweight of the patient. In certain embodiments, the dosage may be in a range of 0.3-2.4, 2.4-4.2, 4.2-6.0, 6.0-7.8, 7.8-10.2, .2-12, 12-14, 14-16, 16-18 or 18-20 mg of antibody or antibody fragment/k gbodyweight of the patient. The dosage of the antibody or antibody fragment wil lremain the same if administered in the form of a bispecific antibody, in combination with another immune checkpoint inhibitor or another antibody or antibody fragment or as an immunoconjugate.
Further, a polypeptide having anti-CTLA4 activity wil lbe administered in the same amounts as the antibody or antibody fragment. 326. 326. id="p-326" id="p-326" id="p-326" id="p-326" id="p-326" id="p-326" id="p-326" id="p-326" id="p-326" id="p-326" id="p-326" id="p-326"
[0326] A singl edose of pharmaceutical formulation of the present invention may contain an amount of the anti-CTLA4 antibody or antibody fragment of the present invention of from about 45 pg of antibody or antibody fragment from about 13,600 mg, or from about 45 pg of antibody or antibody fragment from about 5440 mg. In some embodiments, a singl edose of pharmaceutical formulation of the presen tinvention may contain an amount of the anti- CTLA4 antibody or antibody fragment of the presen tinvention of from to 135 mg to 1,387 mg, or an amount such as 135, 235, 335, 435, 535, 635, 735, 835, 935, 1035, 1135, 1235, 1387 mg. In certain embodiments, the amount of the anti-CTLA4 antibody or antibody fragment of the present invention in a singl edose of the pharmaceutical formulation is in the range of 135-235, 235-335, 335-435, 435-535, 535-635, 635-735, 735-835, 835-935, 935- 1035, 1035-1135, 1135-1235, 1235-1387 mg. The amount of the antibody or antibody fragment in the singl edose of the pharmaceutical formulation wil lremain the same if administered in the form of a bispecific antibody, in combination with another immune checkpoint inhibitor or as an immunoconjugate, or in combination with another antibody or 86WO 2020/092155 PCT/US2019/058066 antibody fragment against another antigen as disclose hereid n. Further, a polypeptide having anti-CTLA4 activity wil lbe included in the singl edose of the pharmaceutical formulation in the same amounts as the antibody or antibody fragment. 327. 327. id="p-327" id="p-327" id="p-327" id="p-327" id="p-327" id="p-327" id="p-327" id="p-327" id="p-327" id="p-327" id="p-327" id="p-327"
[0327] In one example, the anti-CTLA4 antibody or antibody fragment may be conjugated to another immune checkpoint inhibitor molecule or may form part of a bispecific antibody with another immune checkpoint inhibitor. 328. 328. id="p-328" id="p-328" id="p-328" id="p-328" id="p-328" id="p-328" id="p-328" id="p-328" id="p-328" id="p-328" id="p-328" id="p-328"
[0328] The other immune checkpoint inhibitor molecul maye be an antibody or antibody fragment against another immune checkpoint besides CTLA4. The combination can be the anti-CTLA4 antibody or antibody fragment disclose ind this application and the another immune checkpoint inhibitor molecul admie nistered as separate molecules or as a bispecific antibody. Such a bispecific antibody has a binding activity to CTLA4 and a second binding activity to the another immune checkpoint. 329. 329. id="p-329" id="p-329" id="p-329" id="p-329" id="p-329" id="p-329" id="p-329" id="p-329" id="p-329" id="p-329" id="p-329" id="p-329"
[0329] The immune checkpoint may be selected from LAG3, TIM3, TIGIT, VISTA, BTLA, OX40, CD40, 4-1BB, PD-1, PD-L1, and GITR (Zahavi and Weiner, International Journal of Molecular Sciences, vol. 20, 158, 2019). Additional immune checkppoints include B7-H3, B7-H4, KIR, A2aR, CD27, CD70, DR3, and IGOS (Manni et al., Immune checkpoint blockade and its combination therapy with small-molecule inhibitors for cancer treatment, Bbacan, https://doi.Org/10.1016/j.bbcan.2018.12.002, 2018). 330. 330. id="p-330" id="p-330" id="p-330" id="p-330" id="p-330" id="p-330" id="p-330" id="p-330" id="p-330" id="p-330" id="p-330" id="p-330"
[0330] The immune checkpoint is preferably PD-1 or PD-L1. 331. 331. id="p-331" id="p-331" id="p-331" id="p-331" id="p-331" id="p-331" id="p-331" id="p-331" id="p-331" id="p-331" id="p-331" id="p-331"
[0331] It is understood that any of the above formulations or therapeuti cmethods may be carried out using an antibody fragment or an immunoconjugate of the invention in place of or in addition to an anti-CTLA4 antibody. 332. 332. id="p-332" id="p-332" id="p-332" id="p-332" id="p-332" id="p-332" id="p-332" id="p-332" id="p-332" id="p-332" id="p-332" id="p-332"
[0332] Enhancing the host's immune function to combat tumors is the subjec tof increasing interest. Conventional methods include (i) APC enhancement, such as (a) injection into the tumor of DNA encoding foreign MHC alloantigens, or (b) transfecting biopsied tumor cells with genes that increase the probability of immune antigen recognition (e.g., immune stimulatory cytokines ,GM-CSF, co-stimulatory molecules B7.1, B7.2) of the tumor, (iii) adoptive cellula immr unotherapy, or treatment with activated tumor-specific T-cells.
Adoptive cellula imr munotherapy includes isolating tumor-infiltrating host T-lymphocyte s, expanding the population in vitro, such as through stimulation by IL-2 or tumor or both.
Additionally, isolated T-cells that are dysfunctional may be also be activated by in vitro application of the anti-PD-Ll antibodies of the invention. T-cells that are so-activated may then be readministered to the host. One or more of these methods may be used in 87WO 2020/092155 PCT/US2019/058066 combination with administration of the antibody, antibody fragment or immunoconjugate of the present invention. 333. 333. id="p-333" id="p-333" id="p-333" id="p-333" id="p-333" id="p-333" id="p-333" id="p-333" id="p-333" id="p-333" id="p-333" id="p-333"
[0333] Traditional therapies for cancer include the following: (i) radiation therapy (e.g., radiotherapy, X-ray therapy, irradiation) or the use of ionizing radiation to kill cancer cell s and shrink tumors. Radiation therapy can be administered either externall viay externa lbeam radiotherapy (EBRT) or internally via brachytherapy; (ii) chemotherapy, or the application of cytotoxic drug which generally affect rapidly dividing cells; (iii) targeted therapies, or agents which specificall affey ct the deregulated proteins of cancer cell (e.g.,s tyrosine kinase inhibitors imatinib, gefitinib; monoclona lantibodies, photodynamic therapy); (iv) immunotherapy, or enhancement of the host's immune response (e.g., vaccine); (v) hormonal therapy ,or blockade of hormone (e.g., when tumor is hormone sensitive), (vi) angiogenesis inhibitor, or blockade of blood vessel formation and growth, and (vii) palliative care, or treatment directed to improving the quality of care to reduce pain, nausea, vomiting, diarrhea and hemorrhage. Pain medication such as morphine and oxycodone, anti-emetics such as ondansetron and aprepitant, can permi tmore aggressive treatment regimens. 334. 334. id="p-334" id="p-334" id="p-334" id="p-334" id="p-334" id="p-334" id="p-334" id="p-334" id="p-334" id="p-334" id="p-334" id="p-334"
[0334] In the treatment of cancer, any of the previously described conventional treatments for the treatment of cancer immunity may be conducted, prior, subsequent or simultaneous with the administration of the anti-CTLA4 antibodies or antibody fragments. Additionally, the anti-CTLA4 antibodies or antibody fragments may be administered prior, subsequent or simultaneous with conventional cancer treatments, such as the administration of tumor- binding antibodies (e.g., monoclonal antibodies ,toxin-conjugated monoclonal antibodies) and/or the administration of chemotherapeuti agentsc .
F. Articles of Manufacture and Kits 335. 335. id="p-335" id="p-335" id="p-335" id="p-335" id="p-335" id="p-335" id="p-335" id="p-335" id="p-335" id="p-335" id="p-335" id="p-335"
[0335] In another aspect of the invention, an article of manufacture containing an anti- CTLA4 antibody or antibody fragment and other material suseful for the treatment , prevention and/or diagnosis of the disorders described above is provided. The article of manufacture comprises a container and a label or package inser ton or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The containers may be formed from a variety of material ssuch as glass or plastic. The container holds a composition which is by itself or combined with another composition effective for treating, preventing and/or diagnosing the condition and may have a sterile access port (for exampl ethe container may be an intravenous solution bag or a vial having a stopper pierceabl eby a hypodermic injection needle). At least one active agent in the 88WO 2020/092155 PCT/US2019/058066 composition is an antibody or antibody fragment of the invention. The label or package inser t indicates that the composition is used for treating the condition of choice. Moreover, the article of manufacture may comprise (a) a first container with a composition contained therein, wherei nthe composition comprises an antibody or antibody fragment; and (b) a second container with a composition contained therein, wherei nthe composition comprises a further cytotoxic or otherwise therapeutic agent. The article of manufacture in this embodiment of the invention may further comprise a package insert indicating that the compositions can be used to treat a particular condition. Alternatively, or additionally, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other material sdesirable from a commercial and user standpoint, including other buffers, diluents, filters needl, es, and syringes. 336. 336. id="p-336" id="p-336" id="p-336" id="p-336" id="p-336" id="p-336" id="p-336" id="p-336" id="p-336" id="p-336" id="p-336" id="p-336"
[0336] It is understood that any of the above articles of manufacture may include an immunoconjugate of the invention in place of or in addition to an anti-CTLA4 antibody or antibody fragment. 337. 337. id="p-337" id="p-337" id="p-337" id="p-337" id="p-337" id="p-337" id="p-337" id="p-337" id="p-337" id="p-337" id="p-337" id="p-337"
[0337] Finally, the invention also provides kits comprising at least one antibody or antibody fragment of the invention. Kits containing polypeptide, antibodies or antibody fragments, or antibody drug conjugate of the invention find use in detecting CTLA4 expression (increase or decrease), or in therapeutic or diagnostic assays. Kits of the invention can contain an antibody coupled to a soli dsupport, e.g., a tissue culture plate or beads (e.g., sepharose beads). Kits can be provided which contain antibodies for detection and quantification of CTLA4 in vitro, e.g. in an ELISA or a Western blot .Such antibody useful for detection may be provided with a label such as a fluorescent or radiolabel. 338. 338. id="p-338" id="p-338" id="p-338" id="p-338" id="p-338" id="p-338" id="p-338" id="p-338" id="p-338" id="p-338" id="p-338" id="p-338"
[0338] The kits further contain instructions on the use thereof. In some embodiments the, instructions comprise instructions required by the U.S. Food and Drug Administration for in vitro diagnostic kits. In some embodiments, the kits further comprise instructions for diagnosing the presence or absence of cerebrospinal fluid in a sampl ebased on the presenc e or absence of CTLA4 in said sample In. some embodiments, the kits comprise one or more antibodies or antibody fragments. In other embodiments the, kits further comprise one or more enzymes, enzyme inhibitors or enzyme activators. In still other embodiments, the kits further comprise one or more chromatographic compounds. In yet other embodiments, the kits further comprise one or more compounds used to prepare the sample for spectroscopic assay. In further embodiments, the kits further comprise comparative reference material to 89WO 2020/092155 PCT/US2019/058066 interpret the presence or absence of CTLA4 according to intensity, color spectrum ,or othe r physical attribute of an indicator. 339. 339. id="p-339" id="p-339" id="p-339" id="p-339" id="p-339" id="p-339" id="p-339" id="p-339" id="p-339" id="p-339" id="p-339" id="p-339"
[0339] The following examples are illustrative, but not limiting, of the soft gelatin capsules of the present disclosure. Other suitable modifications and adaptations of the variety of conditions and parameters normall yencountered in the field, and which are obvious to those skill edin the art, are within the scope of the disclosure.
EXAMPLES Example 1: Conditionally active biological (CAB) antibodies against CTLA4 340. 340. id="p-340" id="p-340" id="p-340" id="p-340" id="p-340" id="p-340" id="p-340" id="p-340" id="p-340" id="p-340" id="p-340" id="p-340"
[0340] Antibodies against CTLA4 were produced in this Example (Table 2).
Table 2. Conditionally active antibodies against CTLA4 Light Chain Variable Heavy Chain Variable Region Region CTLA4 Antibody BA-087-05-19 SEQ ID NO: 7 SEQ ID NO: 8 BA-087-08-32 SEQ ID NO: 9 SEQ ID NO: 10 BA-087-01-07 SEQ ID NO: 11 SEQ ID NO: 12 BA-087-01-09 SEQ ID NO: 13 SEQ ID NO: 14 BA-087-03-03 SEQ ID NO: 15 SEQ ID NO: 16 BA-087-03-04 SEQ ID NO: 17 SEQ ID NO: 18 BA-087-04-04 SEQ ID NO: 19 SEQ ID NO: 20 BA-087-04-07 SEQ ID NO: 21 SEQ ID NO: 22 BA-087-05-02 SEQ ID NO: 23 SEQ ID NO: 24 BA-087-06-11 SEQ ID NO: 25 SEQ ID NO: 26 SEQ ID NO: 27 SEQ ID NO: 28 BA-087-08-09 BA-087-09-01-03 SEQ ID NO: 29 SEQ ID NO: 30 BA-087-09-01-02 SEQ ID NO: 31 SEQ ID NO: 32 SEQ ID NO: 33 SEQ ID NO: 34 BA-087-09-01-06 BA-087-09-02-02 SEQ ID NO: 35 SEQ ID NO: 36 BA-087-09-02-06 SEQ ID NO: 37 SEQ ID NO: 38 341. 341. id="p-341" id="p-341" id="p-341" id="p-341" id="p-341" id="p-341" id="p-341" id="p-341" id="p-341" id="p-341" id="p-341" id="p-341"
[0341] These anti-CTLA4 antibodies were further characterized. Data for the antibodies BA- 087-05-19 and BA-087-08-32 is presented in this application.
Example 2: ELISA assay for binding activity of anti-CTLA4 antibodies 342. 342. id="p-342" id="p-342" id="p-342" id="p-342" id="p-342" id="p-342" id="p-342" id="p-342" id="p-342" id="p-342" id="p-342" id="p-342"
[0342] The binding activity of BA-087-05-19 and BA-087-08-32 to immobilized recombinan thuman CTLA4 was determined using an enzyme linked immunosorbent assay (ELISA) in pH 6.0 buffer (tumor microenvironment pH) or in pH 7.4 buffer (normal 90WO 2020/092155 PCT/US2019/058066 physiological pH). Seriall dilutey dBA-087-05-19 and BA-087-08-32 were bound to recombinan thuman CTLA4 extracellula domar in immobilized in the well s.The amount of bound BA-087-05-19 and BA-087-08-32 was quantified using anti-human IgG antibody conjugated to horseradish peroxidase (HRP), which then reacted with 3, 3’, 5, 5’ tetramethylbenzidine (TMB) colorimetric substrate to generate a colored product. The OD absorbance in each well was proportional to the amount of BA-087-05-19 and BA-087-08-32 bound. EC50 values at pH 6.0 for binding to human CTLA4 were calculate usingd the nonlinear fit model (variable slope four, parameters )of GraphPad Prism version 7.03. 343. 343. id="p-343" id="p-343" id="p-343" id="p-343" id="p-343" id="p-343" id="p-343" id="p-343" id="p-343" id="p-343" id="p-343" id="p-343"
[0343] EC50 values of the binding activity of BA-087-05-19 and BA-087-08-32 to human CTLA4 at pH 6.0 and pH 7.4 is shown in Tables 3-4 and the binding curves for representative experiments are shown in FIGS. 3A-3B. Both BA-087-05-19 and BA-087-08- 32, show similar binding activities at pH 6.0 and significantly decreased binding activities at pH 7.4 to human CTLA4 when compared to Ipilimumab and an Ipilimumab analogue.
Table 3 Binding activity of BA-087-05-19 and BA-087-08-32 to human CTLA4 at pH 6.0 pti 8.8 ELISA ■Antigen___________________ ituCTLM :"fest Article ؛؛ ;EC® fr-g/mL} 66/27/17 53.04 1068 | ؛ i 9.59 14 59 :EC® (ngSBL) 8809/17 : ؛ 535 ؛ 6.10 ~™™™5tq־־ ™ך" :EC® (ngM)87/tW 1 8.35 ؛ 6.17 7.21 *A 3.03 | ؛ i 1.51-/*8.18 9.78 *A 4.34 :EC® (ng/mL>mean *A $9 $.«*/. 4.27 Table 4 Binding activity of BA-087-05-19 and BA-087-08-32 to human CTLA4 at pH 7.0 pti 7.8 ELISA Aatiges_____________________ iutCTI.A4 :Test Article_________________ ؛ 84.037.93.; 8 98972; BA4؛§?-t>842j،؛SS78؛ Iptlimumab4023624Ipiaaaea. 69900 ;EC® 66/7.7/17 10 95 1118 ؛ ؛ 35.82 cakiMt/d :EC® (ng/mt) 86/29/17 3.71 | 3sS7 I 93.37 not canulated 3.08 ؛ | 50.18 ;EC® lag/mt) 67/11/17 3.($ not calr.iilated :EC® mean *A SO 5.98 *A 4.49 | 6.41 +/■ 3.98 ؛ 58.79*/-29.95 not calculated 344. 344. id="p-344" id="p-344" id="p-344" id="p-344" id="p-344" id="p-344" id="p-344" id="p-344" id="p-344" id="p-344" id="p-344" id="p-344"
[0344] In addition, the binding activity of BA-087-05-19 and BA-087-08-32 to immobilized recombinan tcynomolgus CTLA4 extracellula domar in was also determined by ELISA. The EC50 of the binding activity of BA-087-05-19 and BA-087-08-32 to cynomolgus CTLA4 in pH 6.0 is shown in Table 5. The binding activity of BA-087-05-19 and BA-087-08-32 to cynomolgus CTLA4 at pH 6.0 and pH 7.4 is shown in FIGS. 4A-4B. 91WO 2020/092155 PCT/US2019/058066 Table 5 Binding activity of BA-087-05-19 and BA-087-08-32 to cynomolgus CTLA4 at pH 6.0 Bethod pH ».؛؛£؛.؛ SA Antiges Tess. Articia Ipilimumab t# AAOH?״!: ؛ 86^87-05-19 8A-&87-88-.W 03781 1m ؛ 15.89 ______258.89______ ECSS ? 11.17 ؛ 39.73 292.19 T™2ZS85T1853....
£CS ifig?ml} meas * S3 moPTs-S.....p 345. 345. id="p-345" id="p-345" id="p-345" id="p-345" id="p-345" id="p-345" id="p-345" id="p-345" id="p-345" id="p-345" id="p-345" id="p-345"
[0345] The EC50 of the binding activity of BA-087-05-19 and BA-087-08-32 at a tumor microenvironment pH of 6.0, measured by ELISA were found to be 8.18 ng/mL and 9.78 ng/ml ,respectively, for human CTLA4, similar to the EC50 determined for Ipiliumab and Ipilimumab analogue. BA-087-05-19 has similar binding activity to both human and cynomolgus CTLA4 at pH 6.0, while BA-087-08-32 has a decreased binding activity to cynomolgus CTLA4 compared to its binding activity to human CTLA4 at pH 6.0. The drop in the binding activity of BA-087-08-32 to cynomolgus CTLA4 at pH 6.0 seen in ELISA appears to be specific for the ELISA assay as the same drop was not observe dusing either SPR or FACS. The binding activity of BA-087-05-19 and BA-087-08-32 to either human or cynomolgus CTLA4 at a normal physiological pH of 7.4 measured by ELISA was significantly lower than the binding activities at pH 6.0.
Example 3: pH-dependent binding activity of anti-CTLA4 antibodies 346. 346. id="p-346" id="p-346" id="p-346" id="p-346" id="p-346" id="p-346" id="p-346" id="p-346" id="p-346" id="p-346" id="p-346" id="p-346"
[0346] Binding activity of the antibodies against CTLA4 was tested using an ELISA assay at a range of pH’s from 5.0 to 7.4. Recombinant human CTLA4 extracellula domainr was immobilized in a range of pH buffers in wells (pH 5.0 to pH 7.4), mimicking tumor microenvironment pH (pH 5.5 to pH 6.7) and the normal physiologic alpH (pH 7.4) and the binding activity was measured using ELISA. The antibodies BA-087-05-19 and BA-087-08- 32 were serially diluted and their binding activity to the recombinant human CTLA4 extracellula domainr was measured. The amount of bound antibodies BA-087-05-19 and BA- 087-08-32 was quantified using anti-human IgG antibody conjugated to horseradis h peroxidase (HRP), which then reacted with 3,3',5,5'-Tetramethylbenzidine (TMB) colorimetric substrate to generate a colored product. The OD absorbance in each wel lwas proportional to the amount of BA-087-05-19 and BA-087-08-32 bound. 347. 347. id="p-347" id="p-347" id="p-347" id="p-347" id="p-347" id="p-347" id="p-347" id="p-347" id="p-347" id="p-347" id="p-347" id="p-347"
[0347] The pH inflection point (= 50% of the binding activity at pH 6.0) for BA-087-05-19 was calculate tod be at pH 6.97 with 90% of the binding activity being present at pH 6.66. 92WO 2020/092155 PCT/US2019/058066 The pH inflection point (= 50% to the binding activity at pH 6.0) for BA-087-08-32 was calculate tod be at pH 6.43 with 90% of the binding activity being present at pH 6.2. 348. 348. id="p-348" id="p-348" id="p-348" id="p-348" id="p-348" id="p-348" id="p-348" id="p-348" id="p-348" id="p-348" id="p-348" id="p-348"
[0348] Average OD value s(from 2 replicates) at the different pH’s were plotted against the pH of the buffer using Softmax Pro software (Molecular Devices). Curve fitting was done using the 4-parameter model built into the software .The inflection point of the pH curve (= 50% of the binding activity at pH 6.0) equals parameter C of the fitting equation. Binding activity at pH 6.0 was set to 100%. The pH for 90% binding activity was interpolated from the fitted curve using the "InterpX" function of the Softmax Pro software. 349. 349. id="p-349" id="p-349" id="p-349" id="p-349" id="p-349" id="p-349" id="p-349" id="p-349" id="p-349" id="p-349" id="p-349" id="p-349"
[0349] The average pH’s for 50% and 90% activity for BA-087-05-19 and BA-087-08-32 were calculate usingd the pH values obtained in Experiment s1-4. BA-087-05-19 (lot numbers #6972) and BA-087-08-32 (lot number #6978) were used in Experiments 1-4. Othe r lots of BA-087-05-19 (lot number #6901) and BA-087-08-32 (lot number #6902) were used in Experiment 5. The pH’s for 50% and 90% activity for BA-087-05-19 and BA-087-08-32 determined from the data of Experiment 5 was similar to the average pH value scalculated using the pH values from Experiment s1-4. See Table 6.
Table 6: pH Dependent Binding of BA-087-05-19 and BA-087-08-32 BA-087-08-32 BA4)87D5-1S Antibodies pH (§8%) pH (50%) pH (90%) pH (50%) Experiment 1 !96383817) 6.76 7.04 6,35 6.55■ Experiment 2 (07062817) 6.55 6.99 6. IE 6.37 !87112017) 6.51 6 ,S3 6.17 6.33 Experiment 4 (07112017) 6.43 6,81 7.02 6.1S 6.66 ± 015 6.23 ±0.11 6.43 ± Q.10 average ± SB 6.9 7 ±0.10 Ej^ieriment 5 (06202017) 6,34 6.63 6.2 6.36 350. 350. id="p-350" id="p-350" id="p-350" id="p-350" id="p-350" id="p-350" id="p-350" id="p-350" id="p-350" id="p-350" id="p-350" id="p-350"
[0350] The binding activities of BA-087-05-019 and BA-087-08-32, as wel las positive controls Ipilimumab and Ipilimumab analogue, to recombinan thuman CTLA4 in various pH buffers are shown in FIG. 5. The inflection points of the pH dependent binding for BA-087- 05-19 and BA-087-08-32 were calculate tod be at pH 6.97 and pH 6.43, respectively. 90% of the binding activities for BA-087-05-19 and BA-087-08-32 were presen tat pH 6.34 and pH 6.2, respectively. In addition, weaker binding activities for both BA-087-05-19 and BA-087- 08-32 were detected at a normal physiologic alpH of 7.4 (FIG. 5). 93WO 2020/092155 PCT/US2019/058066 Example 4: Binding kinetics of anti-CTLA4 antibodies 351. 351. id="p-351" id="p-351" id="p-351" id="p-351" id="p-351" id="p-351" id="p-351" id="p-351" id="p-351" id="p-351" id="p-351" id="p-351"
[0351] The binding kinetics of antibodies BA-087-05-19 and BA-087-08-32 were measure d using surface plasmon resonance (SPR) on immobilized recombinan thuman or cynomolgus CTLA4 at pH 6.0 and pH 7.4. The CTLA4 extracellula domainr (human or cynomolgus) was immobilized on the surface of a sensor chip. Different concentrations of BA-087-05-19 and BA-087-08-32 were injected and the binding interactions with the immobilized CTLA4 and a control surface were monitored in real time. The binding kinetics were calculate usingd a 1:1 Longmuir model built into the analysi ssoftware. 352. 352. id="p-352" id="p-352" id="p-352" id="p-352" id="p-352" id="p-352" id="p-352" id="p-352" id="p-352" id="p-352" id="p-352" id="p-352"
[0352] Antibody BA-087-05-19 showed sub-nanomolar binding activity at pH 6.0. The binding activity drops from pH 6.0 to pH 7.4 by about a factor of 2 (Kd [pH 6.0] = 0.5 nM; Kd [pH 7.4] = 1.1 nM). In addition to the lower binding activity at pH 7.4, the SPR signal at pH 7.4 reaches only about 20% of the signal level detected at pH 6.0 indicating that only a smal fractl ion of the BA-087-05-19 that was presen twas able to bind to human CTLA4 at pH 7.4. See Table 7.
Table 7. Binding activity of BA-087-05-19 to human CTLA4 at different pH values pH 6.0 pH 7.4 Kd [s1־] KD [M] Kd [s1־] KD [M] Ka [M-s] Ka [M • s ] 1.34E + 06 6 .31E-04 4.72E-10 9.06E + 05 1.26E-03 1.39E-09 1.26E + 06 6.04E-04 4.81E-10 8.57E + 05 6 . 69E-04 7.81E-10 .98E-04 9.43E-04 1. 10E + 06 5.43E-10 8.44E + 05 1.12E-09 AVG AVG 1.23E + 06 6 .11E-04 4.99E-10 8.69E + 05 9.58E-04 1.10E-09 353. 353. id="p-353" id="p-353" id="p-353" id="p-353" id="p-353" id="p-353" id="p-353" id="p-353" id="p-353" id="p-353" id="p-353" id="p-353"
[0353] BA-087-08-32 also showed sub-nanomolar binding activity at pH 6.0. The binding activity drops from pH 6.0 to pH 7.4 by about a factor of 100 (Kd [pH 6.0] = 0.45 nM; Kd [pH 7.4] = 45 nM). In addition to the lower binding activity at pH 7.4, the SPR signal at pH 7.4 reaches only about 10% of the signal level at pH 6.0 indicating that only a smal fractl ion of the BA-087-08-32 present is able to bind to human CTLA4 at pH 7.4. See Table 8. 94WO 2020/092155 PCT/US2019/058066 Table 8. Binding activity of BA-087-08-32 to human CTLA4 at different pH pH 6.0 pH 7.4 Kd [s1־] KD [M] Kd [s1־] KD [M] Ka [M-s] Ka [M • s ] 3.29E + 06 1.20E-03 3.65E-10 5.04E + 05 2.836E-02 5.25E-08 3.32E + 06 1 .54E-03 4.63E-10 8.59E + 05 8.3E-03 1.49E-08 4.08E + 06 2.14E-03 5.24E-10 1.94E + 05 1.32E-02 6.83E-08 AVG AVG 1.66E-02 3.56E + 06 1.63E-03 4. 51E-10 5.19E + 05 4.52E-08 354. 354. id="p-354" id="p-354" id="p-354" id="p-354" id="p-354" id="p-354" id="p-354" id="p-354" id="p-354" id="p-354" id="p-354" id="p-354"
[0354] Commerically availabl eanti-CTLA4 antibody Ipilimumab (Yervoy™) was used as control under the same conditions and the binding activity was found to be very similar at pH 6.0 and pH 7.4 (Kd [pH 6.0] = 1.39 nM; Kd [pH 7.4] = 1.37 nM). As such, the binding activity of the Ipilimumab did not depend on the pH. See Table 9. The resulting SPR signals are also very similar at both pH 6.0 and pH 7.4.
Table 9. Binding activity of Ipilimumab to human CTLA4 at different pH pH 6.0 pH 7.4 Kd [s1־] KD [M] Kd [s1־] KD [M] Ka [M-s] Ka [M • s ] 9.48E+05 1.39E-03 1.47E-09 1.12E + 06 1.16E-03 1.04E-09 9.47E-04 8.19E + 05 1.16E-09 1.13E + 06 1.52E-03 1.35E-09 7.72E + 05 1.19E-03 1.54E-09 1.17E + 06 2.02E-03 1.72E-09 AVG AVG 8.46E + 05 1 .17E-03 1.39E-09 1.14E + 06 1.57E-03 1.37E-09 355. 355. id="p-355" id="p-355" id="p-355" id="p-355" id="p-355" id="p-355" id="p-355" id="p-355" id="p-355" id="p-355" id="p-355" id="p-355"
[0355] The binding activity of antibodies BA-087-05-19, BA-087-08-32, and Ipilimumab to cynomolgus CTLA4 were tested using the same conditions as described above for human CTLA4. All three antibodies have a fast off-rate and the SPR signal reaches equilibrium at all antibody concentrations tested. The Kd was calculate byd plotting the maximum SPR signal at each concentration against the antibody concentration. Experiment swere run in triplicate at each pH. 356. 356. id="p-356" id="p-356" id="p-356" id="p-356" id="p-356" id="p-356" id="p-356" id="p-356" id="p-356" id="p-356" id="p-356" id="p-356"
[0356] Antibody BA-087-05-19 binds to cynoCTLA4 with a Kd of 1.96 nM at pH 6.0. At pH 7.4 the calculation indicates a Kd > 100 nM. Antibody BA-087-08-32 binds to cynoCTLA4 with a Kd of 5.95 nM at pH 6.0. At pH 7.4 the resulting SPR signal is too low to calculate a 95WO 2020/092155 PCT/US2019/058066 Kd . Antibody Ipilimumab binds to cynoCTLA4 with a Kd of 6.58 nM at pH 6.0 and a Kd of 6.80 nM at pH 7.4.
Example 5: FACS analysis of anti-CTLA4 antibodies 357. 357. id="p-357" id="p-357" id="p-357" id="p-357" id="p-357" id="p-357" id="p-357" id="p-357" id="p-357" id="p-357" id="p-357" id="p-357"
[0357] The binding activities of antibodies BA-087-05-19 and BA-087-08-32 to human and cynomolgus CTLA4 expressed on the cel lsurface of CHO cell ins a pH 6.0 buffer or in pH 7.4 buffer were measured by (FACS). Serially dilute dBA-087-05-19, BA-087-08-32, Ipilimumab and Ipilimumab analogue were added to the CHO cells expressing human or cynomolgus CTLA4. The amount of antibodies bound on the cell wass quantified using anti- human IgG antibody conjugated to fluorophores. EC50 values at pH 6.0 and 7.4 for binding to cells were calculate usingd the nonlinear fit (variable slope, four parameters) model buil tinto GraphPad Prism software (version 7.03). The expression levels of human CTLA4 or cynomolgus CTLA4 on the surface of the CHO cells were determined using a BD QuantiBRITE™ PE kit. 358. 358. id="p-358" id="p-358" id="p-358" id="p-358" id="p-358" id="p-358" id="p-358" id="p-358" id="p-358" id="p-358" id="p-358" id="p-358"
[0358] At least two independent duplicate FACS experiments were performed for each antibody using each of the cel llines. Binding activities of the antibodies to human CTLA4 on CHO cells (CHO-huCTLA4) at pH 6.0 and 7.4 are shown in FIGS. 6A-6B. Binding activities of the antibodies to cynomolgus CTLA4 on CHO cell (CHOs -cynoCTLA4) at pH 6.0 and 7.4 are shown in FIGS. 7A-7B. The binding activities at different concentrations of the antibodies are plotted in these figures. 359. 359. id="p-359" id="p-359" id="p-359" id="p-359" id="p-359" id="p-359" id="p-359" id="p-359" id="p-359" id="p-359" id="p-359" id="p-359"
[0359] The binding activities of BA-087-05-19 and BA-087-08-32 at pH 6.0 measured by FACs were found to have mean ECs0‘s of 350.1 and 243.4 ng/mL, respectively, for human CTLA4, and 316.2 ng/mL and 402.6 ng/mL, respectively, for cynomolgus CTLA4. The binding activities of Ipilimumab and Ipilimumab analogue at pH 6.0 measured by FACs were found to have mean ECs0‘s of 341.1 and 325.4 ng/mL, respectively, for human CTLA4, and 337.5 ng/mL and 319.6 ng/mL, respectively, for cynomolgus CTLA4. The binding activities of BA-087-05-19 and BA-087-08-32 at pH 7.4 were weaker than the binding activities at pH 6.0 even at the highest concentration tested. 360. 360. id="p-360" id="p-360" id="p-360" id="p-360" id="p-360" id="p-360" id="p-360" id="p-360" id="p-360" id="p-360" id="p-360" id="p-360"
[0360] Both BA-087-05-19 and BA-087-08-32 bind to human CTLA4 and cynomolgus CTLA4 with similar affinity as Ipilimumab and Ipilimumab analogue at pH 6.0. However, BA-087-05-19 and BA-087-08-32 each have a much weaker binding activity to human and cynomolgus CTLA4 at pH 7.4 when compared to the binding activities of Ipilimumab and Ipilimumab analogue at pH 7.4. No binding activity was detected for CHO cell thats did not express CTLA4. 96WO 2020/092155 PCT/US2019/058066 361. 361. id="p-361" id="p-361" id="p-361" id="p-361" id="p-361" id="p-361" id="p-361" id="p-361" id="p-361" id="p-361" id="p-361" id="p-361"
[0361] Finally, saturations of the antibodies on the CHO cells expressing human or cynomolgus CTLA4 at pH 7.4 were also measured by FACS (FIGS. 8A-8B). At pH 7.4, less of the antibodies BA-087-05-19 and BA-087-08-32 were bound on the CHO cell thans was the case for the control Ipilimumab analogue.
Example 6: ELISA and FACS analyses of anti-CTLA4 antibody stability 362. 362. id="p-362" id="p-362" id="p-362" id="p-362" id="p-362" id="p-362" id="p-362" id="p-362" id="p-362" id="p-362" id="p-362" id="p-362"
[0362] The binding activities of antibodies BA-087-05-19 and BA-087-08-32 to human CTLA4 at pH 6.0 in buffer and at pH 7.4 in buffer, were measured using different buffers.
Both ELISA and FACS analysi swere used to measure the binding activity. In ELISA analysis, seriall dilutey dBA-087-05-19 and BA-087-08-32 samples were added to human well pre-coates d with CTLA4 and the respective buffer. The amount of bound antibodies was quantified using anti-human IgG antibody conjugated to HRP. The absorbance at 450nm in each measureme ntwas proportional to the amount of antibodies bound. See ELISA data in FIGS. 9A-9F. EC50 value (in ng/mL) for binding to human CTLA4 was determined by absorbance at 450 nm against antibody concentration with Prism variable slope of four- parameter dose-response curve, which was calculated using the nonlinear fit (variable slope , four parameters )model buil tinto GraphPad Prism software (version 7.03). The EC50 values for binding to human CTLA4 as measured by ELISA in different buffers are given in Table s -11. The buffers tested included His buffer (His), Tris buffer (Tris), Glutamine buffer (Glu) and no buffer.
Table 10. EC50 of BA-087-05-19 in different buffers determined by ELISA ؛ « A-037-0:i ?2 ):C50 (גוש/זמ: 19 (69?> His) 8A-D87.-6S-39<5472 •ns) : 8/v(-87-05--34 (09 5־072( : pH 6.0 ؛ pH 74 : 0)3 &،• pH 7.4 TA 5.913 ؛ 4237 ؛ ; 15.41 j 54 36 10.39 110 ؟ 6.25S i 54.55 j 7533. 30.14 : 30.54 ؛ 6.258 ؛ $4.55 T?.W : 7.042 2.(6?. 21.95 Table 11. EC50 of BA-087-08-32 in different buffers determined by ELISA 363. 363. id="p-363" id="p-363" id="p-363" id="p-363" id="p-363" id="p-363" id="p-363" id="p-363" id="p-363" id="p-363" id="p-363" id="p-363"
[0363] In FACS analysis, serially dilute dBA-087-05-19 and BA-087-08-32 samples were added to cell expressis ng human CTLA4. The amount of bound antibodies was quantified using anti-human IgG antibody conjugated to fluorophores The. MFI in each reaction was 97WO 2020/092155 PCT/US2019/058066 proportional to the amount of antibodies bound. The binding activities as measured by FACS are shown in FIGS. 10A-10F. The EC50 value (in ng/mL) for binding to human CTLA4 on cells was determined by MFI of the single tspopulation against antibody concentration with a Prism variable slope of four-parameter dose-response curve, which was calculate usingd the nonlinear fit (variable slope, four parameters )model built into GraphPad Prism software (version 7.03). The EC50 values for binding to human CTLA4 on CHO cells are given in Tables 12-13.
Table 12. EC50 of BA-087-05-19 in different buffers determined by FACS Table 13. EC50 of BA-087-08-32 in different buffers determined by FACS "'V'" ECSO'-wlrdJ 3 84-08705-35 [69?< Uis' 8^C87<18-32 <8978 T: is) 34083-05-32(6978) 3AO87-08-32 (6973 _____ _____ ____ ____ pH74_____ __ ؛__ _____ ^!6.0____ j_____ ££2^ ^^!6.0____ ، ---------------------------------------------- TO ؛ 159 : 848.5 152.5 ؛ 1259 149.6 813.5 158.4 887.5 IzW ؛ 548.8 ؛ 3.179 353.6 : 154-3 176/. 34!;? 358-4 ."675 Example 7: In silico Immunogenecity Analysis of BA-087-05-19 364. 364. id="p-364" id="p-364" id="p-364" id="p-364" id="p-364" id="p-364" id="p-364" id="p-364" id="p-364" id="p-364" id="p-364" id="p-364"
[0364] This study determined the potential immunogenicity of BA-087-05-19 using an EpiVax’s in silico immunogenicity screening toolkit. The software was accesse dthrough ISPRI, a web-based interactive screening and protein reengineering interface .Using the BA- 087-05-19 variable domain as input, the software was used to rate the immunogenic potential on a normalized scal eand predict the potential ADA response. 365. 365. id="p-365" id="p-365" id="p-365" id="p-365" id="p-365" id="p-365" id="p-365" id="p-365" id="p-365" id="p-365" id="p-365" id="p-365"
[0365] The potential immunogenicity of BA-087-05-19 was analyzed and compared to known antibodies on a normalized scale. The data show that BA-087-05-19 has a Tregitope adjusted EpiMatrix Protein Score of -27.70 and a predicted T-dependent antibody respons eof 1.29%. This predicted low immunogenecity falls within the group of optimal antibodies (with low effector and high Tregitope content).
Example 8: Functional assay of potentiating IL-2 secretion by anti-CTLA4 antibodies 366. 366. id="p-366" id="p-366" id="p-366" id="p-366" id="p-366" id="p-366" id="p-366" id="p-366" id="p-366" id="p-366" id="p-366" id="p-366"
[0366] The functional activities of antibodies BA-087-05-19 and BA-087-08-32 in inducing IL-2 secretion by human lymphocytes stimulated with staphylococcal enterotoxin B (SEB) were determined in this example. Serially dilute dBA-087-05-19, BA-087-08-32, Ipilimumab and Ipilimumab analogue were added to human peripheral blood mononuclear cells (PBMCs) 98WO 2020/092155 PCT/US2019/058066 from normal healthy donors stimulate withd SEB. The ability of the antibodies to potentiate IL-2 secretion in SEB-stimulated human PBMCs was quantified using an IL-2 ELISA kit. 367. 367. id="p-367" id="p-367" id="p-367" id="p-367" id="p-367" id="p-367" id="p-367" id="p-367" id="p-367" id="p-367" id="p-367" id="p-367"
[0367] A total of three independent experiment swere performed .In SEB-stimulate d peripheral blood mononuclea cellr (PBMC) cultures, the addition of BA-087-05-19 and BA- 087-08-32 enhanced IL-2 production over the level observed with the addition of isotype control at the level observe dwith Ipilimumab and Ipilimumab analogue at pH 6.2, as shown in FIG. 11 A. On the other hand, IL-2 production did not increase with the addition of BA- 087-05-19 and BA-087-08-32 at pH 7.4 as shown in FIG. 11B. 368. 368. id="p-368" id="p-368" id="p-368" id="p-368" id="p-368" id="p-368" id="p-368" id="p-368" id="p-368" id="p-368" id="p-368" id="p-368"
[0368] At a concentation of 10 pg/mL, BA-087-05-19 promoted a mean 1.4 fold increase and BA-087-08-032 promoted mean 1.5 fold increase in IL-2 production as compare to the isotype control at pH 6.2, similar to the increase observed with Ipilimumab and Ipilimumab analogue. These resul tsindicate that the functional activities of BA-087-05-19 and BA-087- 08-32 are equivalent to the activity observed with Ipilimumab and Ipilimumab analogue at pH 6.2.
Example 9: Promega® CTLA4 blockade assay for anti-CTLA4 antibodies 369. 369. id="p-369" id="p-369" id="p-369" id="p-369" id="p-369" id="p-369" id="p-369" id="p-369" id="p-369" id="p-369" id="p-369" id="p-369"
[0369] The activity of antibodies BA-087-05-19 and BA-087-08-32 in blocking interactions between human CTLA4 and its ligands (CD80 nad CD87) was determined by use of an in vitro Promega® CTLA4 Blockade Assay. Serially dilute dBA-087-05-19, BA-087-08-32, Ipilimumab and Ipilimumab analogue were added to Jurkat effector cell follos wed by addition of aAPC/Raji cell accordis ng to the vendor’s protocol. The blockade of the interactions between CTLA4 and its ligands resul tsin activation of the IL-2 pathway engineered in the Jurkat effector cells, which was quantified using a Bio-Gio® luciferase assay kit. 370. 370. id="p-370" id="p-370" id="p-370" id="p-370" id="p-370" id="p-370" id="p-370" id="p-370" id="p-370" id="p-370" id="p-370" id="p-370"
[0370] The result sshowed that BA-087-05-19 and BA-087-08-32 were able to block the interaction between CTLA4 and its ligands (CD80/CD87) at a similar level observed with Ipilimumab and Ipilimumab analogue at pH 6.0 as shown in FIG. 12A. In contrast, BA-087- 05-19 and BA-087-08-32 were less effective in blocking the interaction of CTLA4 with its ligands at pH 7.4 as shown in FIG. 12B. These result sindicate that the in vitro functional activities of BA-087-05-19 and BA-087-08-32 are equivalent to the activities observed with Ipilimumab and Ipilimumab analogue at pH 6.0 significantly less than the activities of Ipilimumab and Ipilimumab in the blockade at pH 7.4. 99WO 2020/092155 PCT/US2019/058066 Example 10: FACS assay for ligand blocking by anti-CTLA4 antibodies 371. 371. id="p-371" id="p-371" id="p-371" id="p-371" id="p-371" id="p-371" id="p-371" id="p-371" id="p-371" id="p-371" id="p-371" id="p-371"
[0371] The activities of BA-087-05-19 and BA-087-08-32 for inhibiting the interaction of human CTLA4 with its ligands hB7-l (hCD80) and hB7-2 (hCD86) were assayed by FACS to assess the competitive binding at a fixed concentration of BA-087-05-19 and BA-087-08- 32 to CHO cells expressing human CTLA4 in the presence of different concentrations of hB7-l and hB7-2. The amounts of BA-087-05-19 and BA-087-08-32 bound to CHO- huCTLA4 cells were quantified using anti-human IgG antibody conjugated to fluorophores.
The mean fluorescence intensity (MFI) in each reaction was proportional to the amount of BA-087-05-19 and BA-087-08-32 bound to CHO-huCTLA4 as shown in FIGS. 13A-13B. 372. 372. id="p-372" id="p-372" id="p-372" id="p-372" id="p-372" id="p-372" id="p-372" id="p-372" id="p-372" id="p-372" id="p-372" id="p-372"
[0372] In addition, FACS analysis was used to determine the competitive binding of seriall y dilute dBA-087-05-19 and BA-087-08-32 to CHO cells expressing human CTLA4 at a fixed concentration of hB7-l and hB7-2. The amount of hB7-l and hB7-2 bound to CHO- huCTLA4 cells was quantified using anti-His antibody and anti-mouse IgG antibody conjugated to fluorophores. The MFI in each reaction was proportional to the amount of hB7- 1 and hB7-2 bound to CHO-huCTLA4. The data showed that BA-087-05-19 and BA-087-08- 32 blocke dthe interaction of huCTLA4 with its ligands, hB7-l and hB7-2 at level sismilar to those achieved by Ipilimumab and Ipilimumab analogue (FIGS. 14A-14B). 373. 373. id="p-373" id="p-373" id="p-373" id="p-373" id="p-373" id="p-373" id="p-373" id="p-373" id="p-373" id="p-373" id="p-373" id="p-373"
[0373] The data show that BA-087-05-19 and BA-087-08-32 are able to block the interaction between human CTLA4 and its ligands hB7-l (hCD80) and hB7-2 (hCD86) as efficiently as Ipilimumab and Ipilimumab analogue. Competition FACS analysis was only performed at pH 6.0 because BA-087-05-19 and BA-087-08-32 have very limite dbinding at pH 7.4.
Methods Used in the Examples 374. 374. id="p-374" id="p-374" id="p-374" id="p-374" id="p-374" id="p-374" id="p-374" id="p-374" id="p-374" id="p-374" id="p-374" id="p-374"
[0374] The ELISA assay was performed using the following protocol: 1) Coat ELISA plates with 100 pL of 0.5 pg/mL (06_20_17 and 06_28_17 Experiments) or 1 pg/mL (07_06_17 and 07_ll_17 Experiments )recombinant CTLA4 antigen in carbonate-bicarbonate coating buffer 2) Cover plate swith sealing film and incubate overnight at 4°C 3) Decant plates and tap out residual liquid on a stack of paper towels 4) Wash wells twice by dispensing 200 pL of various pH incubation buffer to the wells according to a sampl emap and completely aspirate the contents ) Add 200 pL of various pH incubation buffer to the well accordings to the sample map.
Cover with sealing film and place the plate onto a plate shaker (set to 200 rpm) for 60 minutes at room temperature 100WO 2020/092155 PCT/US2019/058066 6) Decant plates and tap out residual liquid on a stack of paper towels. 7) Serially dilute test substances in various pH incubation buffers to 250 ng/mL, 100 ng/mL or 25 ng/mL. 8) Add 100 pL/well of dilute dtest substances to the plate saccording to the sample map. 9) Cover with sealing film and place the plate sonto a plate shaker (set to 200 rpm) for 60 minutes at room temperature.
) Decant plates and tap out residual liquid on a stack of paper towels. 11) Wash wells three times by dispensing 200 pL of various pH wash buffers to the wells according to the sample map and completely aspirate the contents 12) Dilute the HRP secondary antibody at 1:2500 in various pH incubation buffers 13) Add 100 pL HRP secondary antibody dilute din various pH incubation buffers to each well according to the sample map. 14) Cover with sealing film and place the plate sonto a plate shaker (set to 200 rpm) for 60 minutes at room temperature.
) Decant plates and tap out residual liquid on a stack of paper towels. 16) Wash wells three times by dispensing 200 pL of various pH wash buffer to the wells according to the sample map and completely aspirating the contents 17) Dispense 50 pL per wel lof the TMB substrate solution into all wells of plates. Incubate at room temperature for 3 minutes. 18) Add 50 pL per well of IN HC1 into all wells of the plates. Read plates at 450 nm using Molecular Device SpectraMax 190 microplate reader. 19) The OD450 nm raw data are measured.
) The average OD value s(from 2 replicates) at the different pH were plotted against the pH of the buffer using Softmax Pro software (Molecular Devices). Curve fitting was done using the 4-parameter model built into the software .The inflection point of the pH curve (50% binding activity) equals parameter C of the fitting equation. Binding activity at pH 6.0 was set to 100%. The pH for 90% binding activity was interpolated from the fitted curve using the "InterpX" function of the Softmax Pro software. 375. 375. id="p-375" id="p-375" id="p-375" id="p-375" id="p-375" id="p-375" id="p-375" id="p-375" id="p-375" id="p-375" id="p-375" id="p-375"
[0375] The surface plasma resonance (SPR) assay wass performed using the following protocol: 376. 376. id="p-376" id="p-376" id="p-376" id="p-376" id="p-376" id="p-376" id="p-376" id="p-376" id="p-376" id="p-376" id="p-376" id="p-376"
[0376] The SPR 2/4 instrument ,SPR Affinity Sensors - Amine Flat ,and Immobilization buffer kit are manufactured by Sirra Sensors. The SPR sensor contains four flow cells (FC1- FC4), each of which can be addressed individuall yor in groups. CTLA4 extracellula domainr 101WO 2020/092155 PCT/US2019/058066 was immobilized in FC2 and FC4, whil eBSA was immobilized in FC1 and FC3 (control surface). 377. 377. id="p-377" id="p-377" id="p-377" id="p-377" id="p-377" id="p-377" id="p-377" id="p-377" id="p-377" id="p-377" id="p-377" id="p-377"
[0377] Immobilizatio nwas done following the protocol suggeste dby vendor: (1) The activator was prepared by mixing 200 mM EDC and 50 mM NHS (Sierra Sensors) immediately prior to injection. The amine sensor chip was activated for 480s with the mixture at a flow rate of 25 pL/min. (2) 25 ug/mL of human CTLA4 in 10 mM NaAc (pH 5.0) was injected to FC2 and FC4 respectively at a flow rate of 25 pL/min for 480 s. The chip surface was deactivated with 1 M ethanolamine-H Cl(Sierra Sensors) running through FC1-4 at a flow rate of pL/min for 480 s. (3) The control surface was activated and deactivated using the same conditions, but without injecting protein. (4) The running buffer was switched to PBST with the required pH before the analyte injections. The instrument was equilibrated with the running buffer for 1 hour before the first analyte injection. (5) All analyte injections were done at 25 pL/min at 25 °C 378. 378. id="p-378" id="p-378" id="p-378" id="p-378" id="p-378" id="p-378" id="p-378" id="p-378" id="p-378" id="p-378" id="p-378" id="p-378"
[0378] BA-087-05-19 was diluted in running buffer (buffer PBST pH 6.0 or 7.4) to 5 ug/mL (34.25 nM), 2 pg/mL (13.70 nM), 1 pg/mL (6.85 nM), 0.5 pg/mL (3.42 nM), 0.2 ug/mL (1.37 nM) and 0 pg/mL (0.0 nM). BA-087-08-32 was dilute din running buffer (buffer PBST pH 6.0 or 7.4) to 5 ug/mL (34.25 nM), 2 pg/mL (13.70 nM), 1 pg/mL (6.85 nM), 0.5 pg/mL (3.42 nM), 0.2 pg/mL (1.37 nM) and 0 ug/mL (0.0 nM). 379. 379. id="p-379" id="p-379" id="p-379" id="p-379" id="p-379" id="p-379" id="p-379" id="p-379" id="p-379" id="p-379" id="p-379" id="p-379"
[0379] 100 pL dilute danalyte BA-087-05-19 or BA-087-08-32 was injected over flow cell s 1 and 2 (or 3 and 4) at a flow rate of 25 pL/min for an association phase of 240 s followed by 360 s dissociation. Repeat 6 cycle sof running analyte according to the analyte concentrations in ascending order. The chip surface was regenerated after each cycle of interaction analysi s by injecting 6 pL of 10 mM glycine (pH 2.0). Each set was run a total of 3 times at the same pH. 380. 380. id="p-380" id="p-380" id="p-380" id="p-380" id="p-380" id="p-380" id="p-380" id="p-380" id="p-380" id="p-380" id="p-380" id="p-380"
[0380] Flow cell 1 (or 3) without immobilized protein was used as control surface for reference subtraction. In addition, data with buffer only as analyte (0 nM analyte) were subtracted from each run. Double subtracted data were fitted with the provided analysis software Analyzer R2 (Sierra Sensors) using a 1:1 binding model A. molecular weight of 146 kDa was used to calculate the mola rconcentrations of the analytes. 102WO 2020/092155 PCT/US2019/058066 381. 381. id="p-381" id="p-381" id="p-381" id="p-381" id="p-381" id="p-381" id="p-381" id="p-381" id="p-381" id="p-381" id="p-381" id="p-381"
[0381] The fluorescence-activated cell sorting (FACS) assay was performed using the following protocol. 382. 382. id="p-382" id="p-382" id="p-382" id="p-382" id="p-382" id="p-382" id="p-382" id="p-382" id="p-382" id="p-382" id="p-382" id="p-382"
[0382] Cell staining to determine surface expression of human or cynomolgus CTLA4 1) Seed 3 x 106 cells to T-75 flasks and culture according to the instructions of vendors. 2) On the day of FACS analysis, remove and discard culture medium. 3) Briefly rinse the cel llaye rwith PBS solution. 4) Add 1.5 mL of Detachin solution to each of the T-75 flasks Wait. until cel llayer is dispersed.
) Add 4.5 mL of culture media for the corresponding cel lline sand resuspend cells by gentl epipetting. 6) Pool the cell ands transfe rthe cell suspension to a 50-mL conical tube. 7) Count the cells with trypan blue staining before centrifugation at 1500 rpm for 5 min at 4°C. 8) Wash the cells once with PBS and transfer 3 x 105 cell intos Eppendorf tube. 9) Add 2 pL of mouse anti-CTLA4 (PE conjugated mouse IgGl )or PE-isotype mouse IgGl in 100 pL of PBS solution with 1% BSA per tube and shake at 100 RPM for one hour on ice.
) Wash cells three times with 150 pL PBS solution. 11) Fix cells with 4% PF A for lOmin at R.T., then wash cells with PBS once. 12) Resuspend cells in 100 pL PBS and analyze the cells on NovoCyte flow cytometer. 383. 383. id="p-383" id="p-383" id="p-383" id="p-383" id="p-383" id="p-383" id="p-383" id="p-383" id="p-383" id="p-383" id="p-383" id="p-383"
[0383] FACS analysis of CHO cells expressing human CTLA4 or cynomolgus CTLA4 using tested antibody. 1) Harvest the cells (as 3.3, steps 1 through 7), wash the cells once with PBS. 2) Resuspend the cells in pH 6.0 or pH 7.4 FACS buffer to 3 x 106 cells/mL. 3) Aliquot 3 x 105 cells in 100 pL pH 6.0 or pH 7.4 FACS buffer in 96-well U-bottom plates. 4) Spin down the cell ands discard the buffer.
) Serially dilute test articles in 3-fold dilutions starting at 10 pg/mL (06-16-17, 06-26-17 and 06-28-17 Experiments for total 8 data points or at 100 pg/mL (07_10_17 experiment for total 11 data points) in pH 6.0 or pH 7.4 FACS buffer. 6) Add 100 pL/well of the dilute dtest articles to cells, gently mix well and incubate on ice with shaking (100 rpm) for one hour. 7) Centrifuge the cells at 1500 rpm for 5 min at 4°C. Wash the cells with 150 pL of pH 6.0 or pH 7.4 wash buffer for two times. 103WO 2020/092155 PCT/US2019/058066 8) Dilute the goat anti-human IgG AF488 antibody 1:300 in pH 6.0 or pH 7.4 FACS buffers. 9) Add 100 pL of the dilute dantibody from step above to the cell ands incubate on ice for 45 minutes ,protected from light.
) Pellet the cell ands wash with 150 pL of pH 6.0 or pH 7.4 wash buffer for three times. 11) Fix cells with 4% PF A dilute din IX PBS for 10 min at R.T., then wash cells with IX PBS. 12) Resuspend the cells in 100 pL of IX PBS. 13) Analyze the cell bys NovoCyte Flow Cytomete rusing Ex488nm/Em530nm. Collect at least 20,000 cells. 384. 384. id="p-384" id="p-384" id="p-384" id="p-384" id="p-384" id="p-384" id="p-384" id="p-384" id="p-384" id="p-384" id="p-384" id="p-384"
[0384] FACS data were analyzed using the nonlinear fit (variable slope four, parameters) model built into GraphPad Prism software version 7.03. 385. 385. id="p-385" id="p-385" id="p-385" id="p-385" id="p-385" id="p-385" id="p-385" id="p-385" id="p-385" id="p-385" id="p-385" id="p-385"
[0385] PROMEGA® CTLA4 blockade assay 1) Transfer a vial of Thaw-and-Use CTLA4 Jurkat effector cells (CS186912) from liquid nitrogen storage to the bench on dry ice. Thaw the vial in a 37 °C water bath until cells are just thawed (about 2 minutes). While thawing gently agitate and visuall inspey ct, do not invert. 2) Gently mix the cel lsuspension in the vial by pipetting up and down 2-3 times, and transfe r0.8 mL to the tube labeled "CTLA4 cells" containing 3.2 mL RPMI+10%FBS. 3) Spin the cell downs at 1500 rpm for 10 min and resuspend in 1 mL RPMI+10%FBS. Mix well, divide the cell suspension into two tubes and spin cells down, wash the pell etonce with either pH 6.0 or pH 7.4 assay media, and then resuspend the cell pell etinto 2 mL of pH 6.0 or pH 7.4 assay media. 4) Immediately dispense 25 pL of CTLA4 Jurkat effector cells into the inner 60 wells of 96- wel lplate according to the layout.
) Add 100 pL of sterile water per well to the unused wells surrounded the sample wells. 6) Make serial dilution of 3 x test article stocks in pH 6.0 or pH 7.4 assay media in duplicates starting at 300 pg/mL to generate 10-fold dilution data points. 7) Dispense 25 pL of serially dilute d3 x test article stocks to the wells containing 25 pL of CTLA4 Jurkat effector cells according to the layout. 8) Transfer a vial of Thaw-and-Use CTLA4 aAPC/Raji cells (CS 186911) from liquid nitrogen storage to the bench on dry ice. Thaw the vial in a 37 °C water bath until cells are just thawed (about 2 minutes). While thawing gently agitate and visuall inspey ct, do not invert. 104WO 2020/092155 PCT/US2019/058066 9) Gently mix cell suspension in the vial by pipetting up and down 2-3x, and transfe r0.8 mL to the tube labeled "aAPC/Raji cells" containing 7.2 mL RPMI+10%FBS.
) Spin the cell downs at 1500 rpm for 10 min and resuspend in 1 mL RPMI+10%FBS. Mix well, divide the cell suspension into two tubes and spin cells down, wash the pell etwith pH 6.0 or pH 7.4 assay media once, and then resuspend the cell pell etinto 4 mL of pH 6.0 or pH 7.4 assay media. 11) Immediately dispense 25 pL of CTLA4 aAPC/Raji cells into the inner 60 wells of assay plate salready containing 50 pL cells and antibody solution. The total assay volum eis 75 pL. 12) Place lid on the plates and incubate the plates for 16 hours at 37°C in 5% CO2 humidified incubator. 13) During the 16-hour induction time, warm Bio-GloTM buffer to ambient temperatur eusing a room temperature water bath, prior to addition to Bio-GloTM substrate.
TM 14) Reconstitute Bio-GloT Luciferase Assay System by transferring one bottle of Bio-Gio TM buffer to the bottle containing Bio-Gio substrate.
) After 16-hour induction, remove assay plates from the CO2 incubator and equilibrate at ambient temperatur efor 15 min.
TM 16) Add 75 pL of Bio-Gio reagent to the inner 60 wells of the assay plates. 17) Incubate plate sfor 5-10 min at ambient temperature. 18) Record luminescenc one the SpectraMax i3X plate reader. 386. 386. id="p-386" id="p-386" id="p-386" id="p-386" id="p-386" id="p-386" id="p-386" id="p-386" id="p-386" id="p-386" id="p-386" id="p-386"
[0386] It is to be understood, however, that even though numerous characteristics and advantages of the present invention have been set forth in the foregoing description, together with detail sof the structure and function of the invention, the disclosure is illustrative only, and changes may be made in detail, especial lyin matters of shape, size and arrangement of parts within the principles of the invention to the full extent indicated by the broad general meanings of the terms in which the appended claims are expressed. 387. 387. id="p-387" id="p-387" id="p-387" id="p-387" id="p-387" id="p-387" id="p-387" id="p-387" id="p-387" id="p-387" id="p-387" id="p-387"
[0387] All documents mentioned herein are hereby incorporated by reference in their entirety or alternativel toy provide the disclosure for which they were specificall rely ied upon. The applicant(s )do not intend to dedicate any disclose embodid ment sto the public, and to the extent any disclosed modifications or alterations may not literally fall within the scope of the claims, they are considered to be part hereof under the doctrine of equivalents. 105

Claims (46)

CLAIMED IS:
1. An isolated polypeptide that specifically binds to CTLA4 protein, said polypeptide comprising: a heavy chain variable region including three complementarity determining regions, said regions having H1, H2, and H3 sequences, wherein: (a) the H1 sequence is GFTFSHYTMH (SEQ ID NO: 1); (b) the H2 sequence is FIX1YX2GNX3KX4X5AX6SX7KG (SEQ ID NO: 2); and (c) the H3 sequence is TGWLGPFDX8 (SEQ ID NO: 3), wherein X1 is S or D; X2 is D, H or I, X3 is N or Y; X4 is Y or I; X5 is Y or E; X6 is D or K; X7 is V or M; and X8 is Y or I; and a light chain variable region including three complementarity determining regions having L1, L2, and L3 sequences, wherein: (a) the L1 sequence is RX9SQX10X11GSSYLA (SEQ ID NO: 4); (b) the L2 sequence is GAFSRATGX12 (SEQ ID NO: 5); and (c) the L3 sequence is QQDGSSPWT ( SEQ ID NO: 6), wherein X9 is A or I; X10 is Y, S or H; X11 is V or G; X12 is V or I.
2. The polypeptide of claim 1, wherein the H2 sequence is selected from FIDYHGNNKYYADSVKG, FISYDGNNKIYADSVKG, FISYDGNNKYYADSVKG, FISYDGNYKYYADSVKG, FISYDGNYKYYAKSVKG, FISYHGNNKYEADSVKG, FISYHGNNKYYADSVKG, FISYIGNYKYYADSMKG, FISYIGNYKYYADSVKG.
3. The polypeptide of any one of claims 1-2, wherein the H3 sequence is selected from TGWLGPFDY and TGWLGPFDI.
4. The polypeptide of claim 1, wherein the heavy chain variable region has an amino acid sequence selected from SEQ ID NOS: 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38.
5. The polypeptide of claim 1, wherein the L1 sequence is selected from RASQHVGSSYLA, RASQSVGSSYLA, RASQYGGSSYLA, RASQYVGSSYLA, and RISQYVGSSYLA.
6. The polypeptide of claim 5, wherein the L2 sequence is selected from GAFSRATGI and
7. GAFSRATGV. 282610claimsversion2 1067. The polypeptide of claim 1, wherein the light chain variable region has an amino acid sequence selected from SEQ ID NOS: 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37.
8. An anti-CTLA4 antibody or antibody fragment comprising the isolated polypeptide of any one of claims 1-7.
9. The antibody or antibody fragment of claim 8, wherein the antibody or antibody fragment has a higher binding affinity to CTLA4 protein at a value of a condition in a tumor microenvironment in comparison with a different value of a same condition that occurs in a non-tumor microenvironment.
10. The antibody or antibody fragment of claim 9, wherein the condition is pH.
11. The antibody or antibody fragment of claim 10, wherein the pH in the tumor microenvironment is in a range of from 5.0 to 6.8 and the pH in the non-tumor microenvironment is in a range of from 7.0 to 7.6.
12. The antibody or antibody fragment of any one of claims 8 – 11, wherein the antibody or antibody fragment has a ratio of binding affinity to the CTLA4 protein at a value of a condition in a tumor microenvironment to a binding affinity to the CTLA4 protein at a different value of the same condition in a non-tumor microenvironment of at least about 1.5:1, at least about 2:1, at least about 3:1, at least about 4:1, at least about 5:1, at least about 6:1, at least about 7:1, at least about 8:1, at least about 9:1, at least about 10:1, at least about 20:1, at least about 30:1, at least about 50:1, at least about 70:1, or at least about 100:1.
13. The antibody or antibody fragment of any of one of claims 8 – 12, wherein the antibody or antibody fragment is a chimeric antibody, a multispecific antibody, or a humanized antibody.
14. An immunoconjugate comprising the antibody or antibody fragment of any one of claims 8 – 13.
15. 10715. The immunoconjugate of claim 14, wherein the immunoconjugate comprises at least one agent selected from a chemotherapeutic agent, a radioactive atom, a cytostatic agent and a cytotoxic agent.
16. The immunoconjugate of claim 15, comprising at least two said agents.
17. The immunoconjugate of any one of claims 15 - 16, wherein the antibody or antibody fragment and the at least one agent are covalently bonded to a linker molecule.
18. The immunoconjugate of any one of claims 15 – 17, wherein the at least one agent is selected from maytansinoids, auristatins, dolastatins, calicheamicin, pyrrolobenzodiazepines, and anthracyclines.
19. A pharmaceutical composition comprising: the polypeptide of any one of claims 1 – 7, the antibody or antibody fragment of any one of claims 8 – 13, or the immunoconjugate of any one of claims 14 – 18; and a pharmaceutically acceptable carrier.
20. The pharmaceutical composition of claim 19, further comprising a tonicity agent.
21. A single dose of the pharmaceutical composition of any one of claims 19 – 20, comprising an amount of the polypeptide of any one of claims 1 – 7, the antibody or antibody fragment of any one of claims 8 – 13, or the immunoconjugate of any one of claims 14 – 18 of about 135 mg, 235 mg, 335 mg, 435 mg, 535 mg, 635 mg, 735 mg, 835 mg, 935 mg, 1035 mg, 1135 mg, 1235 mg, or 1387 mg.
22. A single dose of the pharmaceutical composition of any one of claims 19 – 20, comprising an amount of the polypeptide of any one of claims 1 – 7, the antibody or antibody fragment of any one of claims 8 – 13, or the immunoconjugate of any one of claims 14 – 18 in a range of 135 mg -235 mg, 235 mg -335 mg, 335 mg -435 mg, 435 mg -535 mg, 535 mg - 635 mg, 635 mg -735 mg, 735 mg -835 mg, 835 mg -935 mg, 935 mg -1035 mg, 1035 mg - 1135 mg, 1135 mg -1235 mg, or 1235 mg -1387 mg .
23. 10823. The pharmaceutical composition of any one of claims 19 – 22, further comprising an immune checkpoint inhibitor molecule that is different from the polypeptide of any one of claims 1 – 7 and the antibody or antibody fragment of any one of claims 8 – 13.
24. The pharmaceutical composition of claim 23, wherein the immune checkpoint inhibitor molecule is an antibody or antibody fragment against an immune checkpoint.
25. The pharmaceutical composition of claim 24, wherein the immune checkpoint is selected from LAG3, TIM3, TIGIT, VISTA, BTLA, OX40, CD40, 4-1BB, PD-1, PD-L1, GITR, B7- H3, B7-H4, KIR, A2aR, CD27, CD70, DR3, and ICOS.
26. The pharmaceutical composition of claim 24, wherein the immune checkpoint is PD-1 or PD-L1.
27. The pharmaceutical composition of any one of claims 19 – 26, further comprising an antibody or antibody fragment against an antigen selected from PD1, PD-L1, AXL, ROR2, CD3, HER2, B7-H3, ROR1, SFRP4 and a WNT protein.
28. The pharmaceutical composition of claim 27, wherein the WNT protein is selected from WNT1, WNT2, WNT2B, WNT3, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9B, WNT10A, WNT10B, WNT11 and WNT16.
29. The polypeptide of any one of claims 1 – 7, the antibody or antibody fragment of any one of claims 8 – 13, or the immunoconjugate of any one of claims 14 – 18 or the pharmaceutical composition of any one of claims 19 – 28 for use in a method of treating cancer in a patient with cancer.
30. A kit for diagnosis or treatment, said kit comprising the polypeptide of any one of claims 1 – 7, the antibody or antibody fragment of any one of claims 8 – 13, the immunoconjugate of any one of claims 14 – 18, or the pharmaceutical composition of any one of claims 19 – 28 and instructions for using the antibody or antibody fragment, the immunoconjugate and/or the pharmaceutical composition for diagnosis or treatment.
31. 10931. An anti-CTLA4 antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three complementarity determining regions having amino acid sequences of SEQ ID NOS:39-41 and the light chain variable region comprises three complementarity determining regions having amino acid sequences of SEQ ID NOS:42-44.
32. The antibody of claim 31, wherein the heavy chain variable region has an amino acid sequence of SEQ ID NO:8 and the light chain variable region has an amino acid sequence of SEQ ID NO:7.
33. The antibody of any one of claims 31 – 32, wherein the antibody has a higher binding affinity to CTLA4 protein at a value of a condition in a tumor microenvironment in comparison with a different value of a same condition that occurs in a non-tumor microenvironment.
34. The antibody of claim 33, wherein the condition is pH.
35. The antibody of claim 34, wherein the pH in the tumor microenvironment is in a range of from 5.0 to 6.8 and the pH in the non-tumor microenvironment is in a range of from 7.0 to 7.6.
36. The antibody of any one of claims 31 – 35, wherein the antibody has a ratio of binding affinity to the CTLA4 protein at a value of a condition in a tumor microenvironment to a binding affinity to the CTLA4 protein at a different value of the same condition in a non­ tumor microenvironment of at least about 1.5:1, at least about 2:1, at least about 3:1, at least about 4:1, at least about 5:1, at least about 6:1, at least about 7:1, at least about 8:1, at least about 9:1, at least about 10:1, at least about 20:1, at least about 30:1, at least about 50:1, at least about 70:1, or at least about 100:1.
37. The antibody of any of one of claims 31 - 36, wherein the antibody is a chimeric antibody or a humanized antibody.
38. An immunoconjugate comprising the antibody of any one of claims 31 – 37.
39. 11039. The immunoconjugate of claim 38, wherein the immunoconjugate comprises at least one agent selected from a chemotherapeutic agent, a radioactive atom, a cytostatic agent and a cytotoxic agent.
40. The immunoconjugate of claim 39, comprising at least two said agents.
41. The immunoconjugate of any one of claims 39 – 40, wherein the antibody and the at least one agent are covalently bonded to a linker molecule.
42. The immunoconjugate of any one of claims 40 – 41, wherein the at least one agent is selected from maytansinoids, auristatins, dolastatins, calicheamicin, pyrrolobenzodiazepines, and anthracyclines.
43. A pharmaceutical composition comprising: the antibody of any one of claims 31 – 37, or the immunoconjugate of any one of claims 38 – 42; and a pharmaceutically acceptable carrier.
44. The pharmaceutical composition of claim 43, further comprising a tonicity agent.
45. The antibody of any one of claims 31 – 37, the immunoconjugate of any one of claims 38 – 42 or the pharmaceutical composition of any one of claims 43 – 44 for use in a method of treating cancer in a patient with cancer.
46. A kit for diagnosis or treatment, said kit comprising the antibody of any one of claims 31 – 37, the immunoconjugate of any one of claims 38 – 42, or the pharmaceutical composition of any one of claims 43 – 44 and instructions for using the antibody, the immunoconjugate and/or the pharmaceutical composition for diagnosis or treatment. 111
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Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020214748A1 (en) * 2019-04-18 2020-10-22 Bristol-Myers Squibb Company Ipilimumab variants with enhanced specificity for binding at low ph
JP2023537412A (en) 2020-08-13 2023-08-31 ブリストル－マイヤーズ スクイブ カンパニー Methods of Redirecting IL-2 to Target Cells of Interest
EP4240769A1 (en) * 2020-11-06 2023-09-13 Bristol-Myers Squibb Company Dosing and administration of non-fucosylated anti-ctla-4 antibody as monotherapy
US20220306743A1 (en) * 2021-03-01 2022-09-29 Xilio Development, Inc. Combination of ctla4 and pd1/pdl1 antibodies for treating cancer
EP4361176A1 (en) * 2021-06-25 2024-05-01 Chugai Seiyaku Kabushiki Kaisha Use of anti-ctla-4 antibody
WO2023154799A1 (en) 2022-02-14 2023-08-17 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Combination immunotherapy for treating cancer
TW202426499A (en) * 2022-09-16 2024-07-01 大陸商同潤生物醫藥(上海)有限公司 PH-dependent anti-CTLA4 antibody or antigen-binding fragment

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6981720B2 (en) * 2003-11-07 2006-01-03 Agilent Technologies, Inc. Tapered ceramic GC fitting and assembly
WO2009131702A2 (en) * 2008-04-25 2009-10-29 Dyax Corp. Antibodies against fcrn and use thereof
US20140105914A1 (en) * 2011-03-09 2014-04-17 Antitope Limited Humanised anti ctla-4 antibodies
CN104292334A (en) * 2014-04-25 2015-01-21 河南省健康伟业生物医药研究股份有限公司 Fully human anti-CTLA-4 monoclonal antibody, preparation method and application
WO2016196237A1 (en) * 2015-05-29 2016-12-08 Agenus Inc. Anti-ctla-4 antibodies and methods of use thereof
US20180186863A1 (en) * 2012-04-27 2018-07-05 Bioatla, Llc Modified antibody regions and uses thereof

Family Cites Families (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6984720B1 (en) * 1999-08-24 2006-01-10 Medarex, Inc. Human CTLA-4 antibodies
EP2007423A2 (en) 2006-04-05 2008-12-31 Pfizer Products Incorporated Ctla4 antibody combination therapy
JP6121904B2 (en) 2010-09-08 2017-04-26 ハロザイム インコーポレイテッド Methods for evaluating and identifying or developing conditionally active therapeutic proteins
MX363213B (en) * 2012-08-13 2019-03-15 Regeneron Pharma Anti-pcsk9 antibodies with ph-dependent binding characteristics.
MX2016004736A (en) * 2013-10-18 2016-07-26 Regeneron Pharma Methods and compositions comprising a combination of a vegf antagonist and an anti-ctla-4 antibody.
WO2016036916A1 (en) 2014-09-03 2016-03-10 Bioatla, Llc Discovering and producing conditionally active biologic proteins in the same eukaryotic cell production hosts
JP6917902B2 (en) * 2015-02-13 2021-08-11 ソレント・セラピューティクス・インコーポレイテッド Antibody drug that binds to CTLA4
RU2767357C2 (en) * 2016-06-14 2022-03-17 Ксенкор, Инк. Bispecific checkpoint inhibitors antibodies
KR20230130174A (en) * 2016-08-30 2023-09-11 젠코어 인코포레이티드 Bispecific immunomodulatory antibodies that bind costimulatory and checkpoint receptors
US20180339043A1 (en) * 2017-05-26 2018-11-29 Janux Therapeutics, Inc. Modified antibodies
CN111699195A (en) * 2018-02-02 2020-09-22 肿瘤免疫股份有限公司 Mutant anti-CTLA-4 antibodies with improved immunotherapeutic effect but reduced adverse effects

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6981720B2 (en) * 2003-11-07 2006-01-03 Agilent Technologies, Inc. Tapered ceramic GC fitting and assembly
WO2009131702A2 (en) * 2008-04-25 2009-10-29 Dyax Corp. Antibodies against fcrn and use thereof
US20140105914A1 (en) * 2011-03-09 2014-04-17 Antitope Limited Humanised anti ctla-4 antibodies
US20180186863A1 (en) * 2012-04-27 2018-07-05 Bioatla, Llc Modified antibody regions and uses thereof
CN104292334A (en) * 2014-04-25 2015-01-21 河南省健康伟业生物医药研究股份有限公司 Fully human anti-CTLA-4 monoclonal antibody, preparation method and application
WO2016196237A1 (en) * 2015-05-29 2016-12-08 Agenus Inc. Anti-ctla-4 antibodies and methods of use thereof

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